The microbial ecology of equine laminitis of alimentary origin

Milinovich, Gabriel (2008). The microbial ecology of equine laminitis of alimentary origin PhD Thesis, School of Veterinary Science, The University of Queensland.

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Author Milinovich, Gabriel
Thesis Title The microbial ecology of equine laminitis of alimentary origin
School, Centre or Institute School of Veterinary Science
Institution The University of Queensland
Publication date 2008-09
Thesis type PhD Thesis
Supervisor Assoc Prof Darren Trott
Prof Christopher Pollitt
Dr Paul Burrell
Total pages 169
Total colour pages 16
Total black and white pages 153
Language eng
Subjects 300000 Agricultural, Veterinary and Environmental Sciences
Formatted abstract
Equine laminitis constitutes the most serious foot disease of the horse, often resulting in death or
necessitating euthanasia. The factors responsible for initiating laminitis are well described, the most
common being alimentary carbohydrate overload, however, the specific mechanisms by which this
exposure to excess dietary carbohydrates results in lamellar separation remain to be elucidated.
Alimentary carbohydrate overload is characterised by significant population shifts in the equine
hindgut microbiota and the microbial events occurring in the hindgut during the developmental
phase of laminitis are generally accepted to be the source of the trigger factor(s) responsible for
inducing the lamellar separation in the foot. This thesis aims to determine the microbial events
occurring in the equine hindgut during the developmental phase of carbohydrate-induced laminitis.

Using the oligofructose induction model, laminitis was initially induced in five horses and bacterial
populations monitored throughout the development of the disease using a novel, habitat-simulating
medium designed to select for hindgut bacteria with the ability to utilise oligofructose, termed
oligofructose-utilising organisms (OUOs). Culture indicated streptococci, most closely related to
Streptococcus infantarius subsp. coli (synonym: Streptococcus lutetiensis, previously:
Streptococcus bovis), to be the predominant, culturable OUO present in the equine hindgut prior to
the onset of laminitis, while post-laminitis, Escherichia coli became established as the predominant,
culturable OUO. A number of other species which exhibited the ability to utilise oligofructose were
recovered post-laminitis including Enterococcus spp., Bacteroides spp. and Lactobacillus spp.

DNA sequence data produced from the culture-based study was used to design/select fluorescence
in situ hybridisation (FISH) probes targeted to the organisms identified by this and other studies as
having possible roles in the development of equine laminitis. Semi-quantitative FISH was
conducted on paired caecal fluid and faecal specimens collected from four caecally-fistulated horses
at 2-4 hour intervals during the development of laminitis. FISH consistently demonstrated equine
hindgut streptococcal species (EHSS) to proliferate markedly prior to laminitis development, often
reaching relative frequencies in excess of 80% of the total bacteria. Furthermore, marked increases
in EHSS were observed to occur in caecal fluid within 2 hours of oligofructose administration.
FISH targeted specifically to Lactobacillus spp., Enterococcus spp., Allisonella histaminiformans,
Mitsuokella jalaludinii, Clostridium difficile, Bacteroides fragilis and Enterobacteriaceae, all
organisms previously hypothesised to be involved in the development of laminitis, demonstrated
that these groups were either not present or do not establish significant populations in the hindgut
prior to the onset of laminitis. The final study in this thesis aimed to monitor the changes in equine caecal microbiota during the
developmental phase of laminitis using culture-independent methodologies. Serial caecal fluid
specimens obtained from four caecally-fistulated horses were analysed by denaturing gradient gel
electrophoresis (DGGE) of the PCR amplified V2/V3 variable regions of 16S rRNA genes and the
DGGE-determined bacterial populations of interest were subsequently characterised by quantitative
real-time PCR (qPCR) and FISH. Furthermore, caecal fluid samples were analysed for volatile fatty
acid (VFA), lactate and fructo-oligosaccharide concentrations, allowing changes in bacterial
populations to be linked to the production of fermentation end products, substrate (oligofructose)
utilisation and the development of laminitis. The results indicated EHSS proliferation to coincide
with a rapid decrease in caecal VFA concentrations and a marked increase in lactate production
upon entry of oligofructose into the caecum. Furthermore, discrepancies in FISH and qPCR results
provided evidence that EHSS undergo a lytic event, en masse, during the developmental phase of
laminitis. These data suggest that laminitis may be initiated by cellular components liberated from
EHSS following rapid cell lysis.

This thesis constitutes, to date, the most comprehensive study of the microbial ecology of the
equine hindgut during the development of carbohydrate-induced laminitis. It conclusively
demonstrates EHSS, the predominant OUO of the equine hindgut, to proliferate in response to
carbohydrate administration before rapidly decreasing in number, most probably following a lytic
event, further implicating this group of organisms in the pathophysiology of equine laminitis. The
method by which these hindgut bacteria induce lamellar separation remains to be elucidated. This
thesis, however, provides the foundations on which to form subsequent studies to determine the
pathophysiological mechanisms linking the changes in hindgut microbial populations with
pathology in the foot.
Keyword Equine, laminitis, hindgut, microbiology, microbial ecology, Streptococcus, oligofructose.
Additional Notes 1, 25, 27-31, 35, 68, 83-84, 105-107, 149-150, 159

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Created: Fri, 21 Nov 2008, 07:05:37 EST by Mr Gabriel Milinovich on behalf of Library - Information Access Service