MicroRNAs as a novel approach to parasitic nematode control (PhD)


This research shows a novel approach to the problem of controlling H. contortus. MicroRNAs are a group of small non-coding RNA. These molecules can interfere with the production of proteins within cells and cause a reduction in the production of specific proteins. Our results show that four microRNAs are present in the H. contortus genome as part of a cluster called the Hco-miR-5352 cluster. This cluster is found in a range of different parasitic nematodes which are all found in the gastrointestinal tract and is highly expressed in the adult stages of H. contortus. This suggests that this microRNA may have a role in parasitism. The excretory/secretory (ES) products of parasitic helminths are a known source of products that can suppress the host immune response. The project focused on characterising the microRNA content of the ES and identified that the amount of microRNA in the adult worms compared to the microRNAs in the ES is different. This difference suggests that the H. contortus can control what microRNAs is released into the ES. Furthermore, microRNAs in bodily fluids such as blood often exist within extracellular vesicles to protect the microRNA from being destroyed and can transfer microRNAs from cells to cells. We were also able to identify these extracellular vesicle-like structures which we showed also contain certain microRNAs.
These H. contortus specific microRNAs were also identified in H. contortus infected sheep abomasal tissues showing that these microRNAs are released in vivo and strongly suggesting that the target of this microRNA is a sheep gene. To investigate the possible interaction of the parasitic microRNA Hco-miR-5352 and sheep genes, several different experiments were attempted to elucidate the target of Hco-miR-5352. Mouse cells exposed to Hco-miR-5352 resulted in significantly decreased expression of over 300 genes, the vast majority of which were immune related.
The work presented here suggests a potential role for microRNAs in modulating the host immune system. If so, these microRNAs could be used as a drug target. In various human diseases such as cancer, microRNAs have been identified in a range of different body fluids and have been shown to have potential as biomarkers of infection, such as in cancer and heart disease. Preliminary experiments were unable to amplify H. contortus miRNAs from the serum of infected sheep, although recent work has shown that microRNAs can also be detected in human faeces and used as a non-invasive biomarker for colorectal cancer. Perhaps the most interesting aspect of these ES microRNAs is the possible transfer of microRNAs between the nematode and the host with the subsequent targeting of host mRNAs. This cross-species transfer of microRNAs has been shown in other nematodes and plant microRNAs and raises the possibility of utilising extracellular vesicles to deliver microRNAs to specific cells to modulate the expression of particular genes or as a target to reduce infection.
In conclusion, this project has shown that certain microRNAs are conserved in gastrointestinal nematodes. These microRNAs can also be detected in the ES products of H. contortus, either in extracellular vesicles or the supernatant and these microRNAs can be detected in sheep tissue at the site of infection. Studies on the function of H. contortus microRNAs suggests a possible role in modulating the sheep immune system although further studies will be required to elucidate the direct targets of Hco-miR-5352 and its contributions to H. contortus survival in vivo.
Beef & Lamb
Project code:
01 October 2012 - 30 September 2015
Hybu Cig Cymru - Meat Promotion Wales (HCC), Quality Meat Scotland
AHDB sector cost:
Total project value:
Project leader:
University of Glasgow


61110028 Final Report Oct 2016

About this project

The Challenge

Gastro-intestinal (gi) nematodes of ruminants are a major financial and welfare issue world-wide, including the UK. At present, control is based upon the use of anthelmintics, a situation that is threatened by the continued spread of anthelmintic resistance. We have adopted a novel approach to the control of H. contortus by focusing on defining the function of microRNAs in this important parasite. miRNAs are small non-coding RNAs that regulate the expression of other genes and are essential for the correct development of free-living nematodes such as Caenorhabditis elegans. Our recent results suggest that, in addition to functioning in development, parasite miRNAs may play key roles in modulating host immune responses. We have cloned multiple miRNAs from H. contortus (1) and shown that one of these is abundantly expressed in adult worms, is highly conserved in other gi nematodes and is predicted to regulate the expression of a mammalian gene involved in the activation of T cells. We will investigate the potential of parasite miRNAs to influence infection outcome, specifically by altering the balance of T cell responses.

Many parasitic nematodes produce excretory-secretory (ES) products that suppress host immune responses and that likely interfere with the ability to vaccinate (2). Almost all studies to date have characterised protein components of the ES, but as miRNAs are released by eukaryotic cells and are able to traffic from one cell to another (3), we propose that secreted miRNAs may represent a novel mechanism by which parasitic nematodes down-regulate potentially protective immune responses. Thus the aim of this project will be to characterise miRNAs in the ES of the ovine abomasal nematodes H. contortus and T. circumcincta and determine how these small molecules interact with components of the host immune response to regulate the development of immunity. The outcomes will have important relevance to improving parasite control not only in sheep but also in other ruminant hosts.

The Project

This proposal will adopt a novel approach to the problem of parasitic nematode control in ruminants. Specifically, we will study the role of microRNAs (miRNAs) from the gastro-intestinal parasites Haemonchus contortus and Teladorsagia circumcincta in modulating host immune responses. We will investigate whether inhibiting parasite miRNA function enhances protective immune responses and may offer a novel strategy for nematode control

Adult parasites recovered from donor sheep at post-mortem will be cultured in vitro and ES collected. miRNAs will be precipitated from spent culture medium using standard conditions and characterised by probing a microarray (already available) containing all 192 H. contortus miRNAs identified to date. This analysis will give an overview of all miRNAs secreted by H. contortus. Based on our initial analysis, we anticipate that at least some of the key miRNAs will be conserved in T. circumcincta and identified by our microarray approach. In addition, we will identify selected miRNAs in spent tissue culture medium by RT-PCR using gene-specific primers. 

Subsequent experiments will investigate the interaction of miRNAs with components of the host immune response, starting initially with T cells. We will address the following questions: do parasite miRNAs interfere with activation, proliferation or cytokine production by T cells? Do specific miRNAs regulate the expression of mammalian immune molecules? What is the potential outcome of this regulation on immune responses to the parasite?

These initial experiments will be carried out in vitro using well-characterised approaches and are important to define the experimental parameters for subsequent studies aimed at determining whether miRNAs released in vivo at the mucosal interface affect T cell function. This part of the project will be carried out with Dr Tom McNeilly at the Moredun Research Institute (MRI) with whom we have excellent collaborations. Here we will study the effect of gi nematode infection on aspects of the ruminant immune response using the natural host, the sheep. We will examine whether immune molecules shown to be regulated by miRNAs in vitro are similarly regulated by H. contortus and T. circumcincta infection in vivo, by analysis of archived tissue and abomasal T cells from infected sheep using immunochemistry, qRT-PCR and FACS analysis with appropriate markers/probes; we will seek to ascertain the effect of this immune regulation on the outcome of infection by studying the polarisation of T cell responses in the abomasum; finally we will investigate the potential of locked nucleic acid inhibitors (LNA-miRNAs) to neutralise the activity of secreted miRNAs both in vitro and in vivo. These small molecules are very stable in serum and have been shown to inhibit miRNA function in vivo in other disease models (4), indicating their potential as novel therapeutic agents. Blocking miRNA function may allow a protective immune response to develop and improve vaccination and infection outcome. Thus this project has the potential to shed new light upon the mechanisms by which parasitic nematodes modulate immune responses and offer a novel treatment strategy to control these important parasites.



Henry Gu