Mast cell recruitment and activation as measures of cyathostomin burden
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Date
04/07/2015Author
Clements, Ruth Jocelyn Muriel
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Abstract
Cyathostomins are potentially life threatening parasitic nematodes of adult horses
and are highly prevalent worldwide. Infected animals may be asymptomatic or show
clinical signs of weight loss, diarrhoea and colic. Third and fourth stage larvae spend
a large proportion of their lifecycle encysted in the large intestinal wall where they
cannot currently be detected ante mortem. Mast cells are commonly found at
interfaces to the external environment, such as the rectum, and these cells and the
proteinases they produce have been implicated in protective host immune responses
against nematode infection in animals. Previous studies have demonstrated an
increase in caecal mast cell proteinase expression during cyathostomin infection.
Prior to this study, there were two known equine mast cell proteinases, which had
been purified and characterised from a mastocytoma (equine tryptase [eqTRYP] and
equine mast cell proteinase-1 [eqMCP-1]). However, as many mammalian species
express multiple closely-related chymases it was hypothesised that other equine mast
cell proteinases exist that have not yet been characterised and which may be more
closely associated with the level of worm burden. The primary objective of this study
was to investigate the recruitment of mast cells to the large intestine in cyathostomin
infected horses and the expression of mast cell proteinases in response to infection. A
further aim was to evaluate the potential of associated mast cell proteinase assays or
rectal biopsy mast cell enumeration for utility in diagnostic tests to estimate
cyathostomin mucosal burden. A secondary objective was to explore the existence of
further mast cell proteinases and the relationship of these enzymes to cyathostomin
mucosal burden. Optimised sampling protocols, parasitological, histological and
immunohistochemistry techniques were performed to enumerate cyathostomin
mucosal burden and to characterise the mast cell populations in the caecum, right
ventral colon (RVC) and rectum of naturally infected horses (n=28). Mast cell
populations correlated throughout the intestine, providing further evidence of the
common mucosal system. EqMCP-1 and eqTRYP labelled mast cells were identified
throughout the large intestine. Significant positive linear relationship existed between
rectal proteinase-labelled mucosal mast cell populations and both the combined total
cyathostomin mucosal burden (CTMB; eqMCP-1, p=0.018; eqTRYP, p=0.048) and
the combined total luminal burden (CTLB; eqMCP-1, p=0.009; eqTRYP, p=0.007).
Concentrations of eqMCP-1 and eqTRYP in (i) serum, (ii) local serum from venous
blood draining the large intestine, and (iii) large intestinal tissue homogenates were
assessed using ELISA. There was no significant correlation identified between local
and peripheral serum proteinase concentrations suggesting that peripheral serum
proteinase levels are not representative of the local proteinase response. There was
however a significant negative relationship between peripheral serum eqMCP-1
concentrations and the CTMB, which could relate to the activation and sequestering
of proteinases within the gut lumen. Concentrations of eqMCP-1 and eqTRYP
measured in local serum did not significantly positively correlate with cyathostomin
mucosal burden. There was a significant association observed between intestinal
tissue levels of eqMCP-1 and eqTRYP and the CTMB in the RVC (p<0.023),
providing support for their role in the immune response. Four proteinase sequences,
equine tryptase (TLP1), Granzyme B-like (GZMBL), putative equine Mast Cell
Proteinase-1 (CLP1) and Granzyme(BGH)-like (GZM(BGH)L), were sequenced and
the local transcription levels of each of these enzymes assessed using quantitative
reverse-transcription PCR. The expression of TLP1 was closely correlated with
GZMBL expression, and there was a significant positive relationship observed
between TLP1 and GZMBL transcript levels and combined total mucosal burden in
the RVC. Both GZM(BGH)L and CLP1 transcript levels were also positively
correlated with each other, but the levels of these transcripts were not statistically
correlated to any of the cyathostomin parasitological measures assessed here.
This work has provided the basis for further rectal biopsy studies to examine the
important dynamics of the mast cell response to cyathostomin infection. The results
from this thesis, with the demonstration of novel proteinases, are encouraging for
further investigation into equine mast cell proteinases and their role in cyathostomin
infections.