Mast cell recruitment and activation as measures of cyathostomin burden
Clements, Ruth Jocelyn Muriel
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.