Horse gut microbiome
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Transcript of Horse gut microbiome
POST-SURGICAL GUT MICROBIOMEUnderstanding intestinal dysfunction
Gut microbiotaMore than 1014 resident bacteria in humanGITThere is a life-long bidirectional and symbiotic relationship with the hostGut microbiota: the total microbial ecosystemGut microbiome: the collective genomic content;150 times the human gene pool
Importance: digestionAnaerobic microbial fermentation of fibers in hind gutThe volatile fatty acids, the main end products, supply
75%-80% of the absorbed energy from poor quality hay20%-30% of the total energy requirements of a horse
Importance: normal development of immune system
Importance: intestinal barrierGerm free mice compared to conventional mice showed
Impaired intestinal morphology, cell renewal propertiesDecreased total intestinal surface areaAberrant intestinal morphology
Shorter ileal villiSmall intestinal crypts
Lower rate of ileal and Peyer's patches turn over8 days after colonization cell renewal reverted to the same degree of conventional mice
Importance: intestinal barrier
Germ free mice showed also:
Reduced level of antimicrobial peptides secreted by gut epitheliumLower number of mucin-secreting goblet cellsThinner less stable and compact mucin layer
Importance: Health and diseaseReduced bacterial biodiversity (dysbiosis)
In obese compared to lean individualsIn infants with colic compared to those without colic
Gut microbial ecosystem differs between children with or without atopic eczemaWith or without autism
probiotic or prebiotic supplementation to liver disease patientsIncreased intestinal stability (reduced permeability)Decreased bacterial translocation to the liverAmeliorated symptoms of hepatic encephalopathy due to decreased ammonia production
Importance: health and diseaseMarked differences of gut microbita between horses with colitis compared with normal horsesHorses fed diets rich in readily fermentable carbohydrate are:
at increased risk of developing laminitis, simple colonic obstruction and distension with concurrent changes in hind gut bacterial population compared with forage fed animals
Methods to study gut microbiota
Microbita study: Culture based techniques
Isolation and culture on selective and non-selective mediaidentification through:
colony morphological characters, growth patterns on different medias, biochemical characters
Culture based techniques: pros and cons
ProsRelatively inexpensiveWidely available, Possible physiological and biochemical studies, Can provide a good indication of ecosystem complexity
ConsMost of the bacterial community is unculturable
only 10% of direct microscopic count from small intestine and 30% from caecum and colon of horse are culturable60-70% of human gut bacteria are not culturable
methods of sampling, transport, cultivation and identification are inconsistent among the different studies; results discrepancyvery laborious and time consuming
Study Method
sample M.Os
Number (log10 CFU/g)*
Studied effect Result
Medina et al., 2002
Culture
Colonic content
Total anaerobesLactobacillistreptococci
8.86.96.9
Reliability of saccharomyces cerevisiae to limit the negative impact of concentrate overload
The decrease in pH and the increase in lactic acid production were ameliorated
Milinovich et al., 2008
qPCRCecal content
Total bacteriaEHSS
1210
Changes in cecal microbiota in response to oligofructose induced laminitis
Drastic increase in quine hindgut streptococcal species count
Culture vs. molecular based techniques: horse’s gut
* log number of the colony forming units per gram of sample (faeces or gut content)Total bacterial counts obtained by culture-based methods are many logs less than the results of qPCR
Molecular-based techniques
Samples to study: Feces Vs. intestinal content
A non-invasive surrogate for the lower intestinal microbiota Human Microbiome Project, 2012.Reflect the shifts in caecal microbiome in horses with experimentally induced laminitis yet, the relative abundance is not reflected Milinovich et al., 2007.Represent the microbial diversity of the distal colon (dorsal colon) Hastie et al., 2008.
Fecal samples preservationNo difference in microbial composition between samples that processed directly after defecation and those stored for 24 hours at room temperature Carroll et al. (2012.long-term storage of faecal samples at -80 for up to 6 months has no detrimental effects on microbial community structure and diversity Wu et al. (2010.
Microbita study: Molecular-based techniques basis
The 16s rRNA gene constitute the fundamental basis of molecular studies of microbial communities
50S
30S
23S
5S
16s
Plus 32 proteins
Plus 21 proteins70S
RNA
16s rRNA: Reliable phylogenetic marker
homologous function and universal location in all micro-organismsContains conserved regions; did not change over timeUnique nine variable regions (v1-v9). universal primer can be used to amplify nearly all rRNA genes of all microorganismsHas appropriate length (about 1.5 kbp) compared to 5S (short) and 23S (long) rRNA molecules.
Part of 16s rRNA gene
Microbiota studies: Overview of techniques to characterize the gut microbiota
Faecal sample FISH
DNA isolation
Amplification of 16s rRNA gene by PCR
Separation of 16srRNA (DNA Finger
printing)Band
resolution
Microbiome shotgun
sequencing
Direct sequencing of 16S rRNA amplicons
Sequencing of cloned 16s rRNA amplicons
Quantitative PCR
DNA microarrays
Band excision and sequencing
Probe hybridization
Culture
Extraction of DNA and amplification of 16S rRNA genes
Fraher, M. H. et al. (2012)
Molecular techniques: FISH
Fluorescence in situ hybridization
Molecular techniques: DNA microarray
DNA Microarray
DNA finger printing: DGGE, TGGE
Conventional electrophoresis
DNA fingerprinting
454 pyrosequencing
T. Liloglou, 17 Jul2013
Pyrosequencing
39% 36% 37% 32% 39% 36% 38% 40% 43%
EE SS AA TT CC GG TT5
GG AA TT TT CC10
TT GG TT CC GG15
TT GG TT GG CC20
TT TT AA GG TT25
CC TT GG TT CC30
AA GG TT CC TT35
GG TT CC GG TT40
GG TT AA GG TT45
CC TT GG TT CC50
GG GG TT0
10
20
30
40
50
60
A2 : Y GGGT AT T T T YGY GT GGTGTTTTGYGGTYGTYGTYGTTGTGGTYGTTYGGGGTGGGGTGTGAGGAGGGGA
T. Liloglou, 17 Jul2013
3’ – TGTCGAGTCAGGTAAATTTTGAAAGCCA – 5’ 5’ - ACAGCTCAG
Pyrosequencing: Principle
TCCA CTTTAAAA TTTCGGT
4×3×2×1×
T C A CT A T C G T
Dispensation Order Sequence
454 Sequencing vs. other techniquesProvided an open ended view of microbial communitiesNon sequencing techniques: only known bacterial groups can be detected using pre-designated oligonucleotide probes.Sequences can't be directly assigned to speciesSequences are annotated into operational taxonomic units (OTU) (phylotypes)OUT includes cluster of similar sequences with identity threshold of 97%
Pitfalls of molecular-based techniques: Extraction bias
Rigorous processing; excessive fragmentation of bacterial genome; formation of more chimeric DNAThe different extraction methods have resulted in
extraction of different quantities of DNAvariation in phylotype abundance and composition of microbial communities
Pitfalls of molecular-based techniques: Inhibitors
Inhibitory substances in sample; can inhibit the PCR amplification of the extracted DNA:
Bile salts and complex polysaccharides in faecesCollagen in foodHeme, immunoglobulin g and lactoferrin in blood Humic acid in soilMelanin and myoglobin in tissues polysaccharides in plants; proteinases and calcium ions in milkurea in urine
Pitfalls of molecular-based techniques: Amplification bias
Differential or preferential PCR amplificationFormation of PCR artefacts (chimeric molecules, deletion mutant, and point mutant)DNA contamination (false positive) 16s rRNA sequence variation due to variation in number of rRNA gene regions (rrn operon) Hairpin lope, primer dimers formation,
Pitfalls of molecular-based techniques: Universal primers are not universal
No single set of primers can guarantee amplification of all prokaryotic 16s rRNA genesThe coverage rate of twenty nine known primers including 13 Archaea-specific, 9 Eubacteria-specific, and 7 universal primers based on RDP database.
Average coverage rate of these primers are 85%, 77.4% and 83.3% respectively30.6% of known primers have a coverage rate less than 90%.
Molecular studies of horse’s gut: Colitis vs. normalFirmicutes (68.1% control, 30.3 colitis),Bacteroids (14.2% control, 40.0% colitis) Proteobacteria (control 10.2%, 18.7 colitis),Costa et al., 2012454 pyrosequencing
Horse’s gut: forage vs. concentrate fed and SCOD
Daly et al., 2012: Quantitative oligonucleotide hybridization, Colonic content
Lachnospiraceae
Bacteroides
Bacillus-Lactobacillus-Streptococcus group
Horse’s gut: forage vs. concentrate fed and SCOD
Daly et al., 2012: Quantitative oligonucleotide hybridization, Colonic content
Fibrobacter spp.
Ruminococcaceae
Normal horses vs. those with chronic laminitis
Steelman et al., 20122. Pyrosequencing fecal samples
17 29
Normal horses vs. those with chronic laminitis
Steelman et al., 20122. Pyrosequencing fecal samples
108