CAT Task Module 9 : Summer Research Project assignment
Group 3 / Topic 3 : Dictyocaulus viviparus
This is an individual assignment which will be summatively assessed and which will contribute 10% to your end of year mark for BVMS2.
Section which must be completed and which will be graded
Section which will not be assigned a grade but MUST be completed Section which is completed on your behalf [No input required from you]
Part 1 : Summer research project application form
This form is derived, in parts, from the Wellcome Trust Biomedical Sci ences vacation research application form [2014 form : http://www.wellcome.ac.uk/Funding/Biomedical -science/Funding-schemes/PhD-funding-and-undergraduate-opportunities/WTD004448.htm]
Please tick the scientific area that your application falls under [see Wellcome Trust
http://www.wellcome.ac.uk/Funding/Biomedical-science/Application-information/WTD039181.htm for more information on funding streams]: [1.5 marks]
Infection and Immuno-biology
x
Genetic and Molecular Sciences
Population Health Cellular, Developmental & Physiological Sciences Neuroscience and Mental Health
1. DETAILS OF STUDENT
(a) Title: Do not complete First name and
middle initials: Do not complete Surname: Do not complete (b) E-mail address: Do not complete
(c) Name of University/College: (where full time undergraduate)
Glasgow University
(d) Type and title of degree: (e.g. BVSc, BVMS etc)
BVMS
(e) Date degree course commenced: September 2013 (f) Year of course: (e.g. 2nd year of a 3 year course = 2/3) 2
2. RESEARCH PROJECT TITLE
Title of project: (no more than 220 characters) [2 marks] [provide appropriate title based on project description]
The use of RNA data, gene expression and reverse transcriptase in understanding how irradiated larval vaccine works to produce immunity against Dictyocaulus Viviparus.
3. LENGTH OF PROJECT: (max 8 weeks) 8 weeks
4. PROJECT DESCRIPTION (no more than 500 words) : Background
Lungworm or parasitic bronchitis in cattle is caused by infection with a nematode (roundworm) named Dictyocaulus viviparus. Infection is characterized by coughing, increased breathing rate (tachypnoea) and can be fatal following heavy infection. Animals that recover from infection develop immunity that protects them from subsequent infection and clinical disease. Based on the knowledge that infection can induce protective immune responses, researchers aimed to develop a vaccine to protect cattle against parasitic bronchitis. Following extensive research, a vaccine was developed in the 1960s which proved highly effective in protecting young calves against infection. This is a live attenuated vaccine which comprises of two doses of irradiated larval parasites (infective L3 stage) administered orally to calves before turnout. The irradiated larvae induce protective antibody responses which protect animals from natural infection in the field, but do not cause clinical disease.
Aims and methodology
The irradiated larval vaccine is still used today in the UK and several other countries where lungworm is endemic. This is sold commercially as Huskvac. However, vaccine production requires a supply of infective L3 stage larvae which can only be obtained by keeping animals infected with D. viviparus parasites, collecting eggs from faecal samples and culturing eggs to the L3 stage. This is expensive, labour intensive and has associated ethical and welfare concerns. Ideally, a vaccine incorporating specific parasite anti gens (proteins) that induce similar protective immune responses to the irradiated larval vaccine could be developed to replace the Huskvac vaccine. The overall aim of the proposed project is to understand how the irradiated
larval vaccine works. This knowledge may, in future studies, be applied to design a molecularly defined
vaccine which would be easier and cheaper to produce.
It is speculated that irradiated larvae develop more slowly and show differences in gene expression compared to non-irradiated larvae. These differences may be associated with altered development and induction of protective immune responses by the vaccine. Using RNA sequencing technology, data on specific genes expressed by organisms (the transcriptome) and the level of gene expre ssion can be obtained. This project will use RNA sequence data available for untreated L3 and irradiated L3 of D. viviparus to identify changes in gene expression that may help explain the protection achieved by vaccination.
1. Use available RNA sequence data to identify genes differentially expressed between untreated and irradiated L3 larvae that have been allowed to develop for 48 hours in vitro. The focus will be on genes uniquely expressed or with >2 fold difference in expression level between the tw o larval types
2. Apply bioinformatic tools (NCBI database searches, comparative sequence analysis, predicted protein function) to characterise genes showing differences in expression between untreated and irradiated larvae 3. Confirm differential expression of genes in untreated and irradiated L3 by reverse transcriptase PCR (RT-PCR)
5. What techniques/training will the scholarship provide? (no more than 100 words) [2 marks]
This project will provide training in the use of RNA sequencing technology which will better my understanding of genome structure and allow me to gain an understanding of their composition and function. This could be applied to future research involving genetics. The project uses bioinformatics tools, many of which are online databases, giving information on genetic sequences. The project will teach me to use these databases and give me the ability to characterise genes using these tools. The use of reverse transcriptase PCR will improve my practical lab skills and scientific technique as well as my ability to analyse results.
6. ETHICS & REGULATORY ISSUES [1.5 marks] [refer to BVMS1 module 1 and 2 Welfare and Legislation lectures
and/or web resources]
(a) Does the project involve the use of human participants, biological samples or personal data?
YES NO X
(c) Will this project involve the use of animals or animal tissue? YES X NO (d) If yes, does the proposal include procedures which require a Home Office licence? YES X NO
7. Summary of the skills you would bring to the project (no more than 100 words) [2 marks]
I am an appropriate candidate for the research project because I am focused and precise in my work. I have knowledge from lectures at university on the disease caused by lungworm, vaccination, genetics (including RNA sequencing and PCR) and on immunity which give me a solid understanding of the science behind this project. I work well in a lab environment and I am good at applying theory to practical tasks. Furthermore, am very hard working and work well with other people as well as on my own.
8. SUPPORTING INFORMATION
(a) Please explain how your project relates to the improvement of human or animal health? (no more than 250 words) [5 marks]
The project relates to the improvement of animal health as the results give information that could lead to the development of a vaccine against Dictyocaulus Vivparus. This vaccine would prevent infection and thus prevent parasitic bronchitis which compromises the animals welfare causing coughing, tachypnoea and in some cases death. Currently, infected animals are kept to produce larvae for the irradiated vaccine. This is a compromise of their welfare as they are kept for a prolonged amount of time, suffering from the disease. These animals will no longer need to be kept and so will no longer suffer.
This is also a cheaper way of producing the vaccine so more farmers may use it and more animals will be treated. It would also mean that the farmer would have more money to spend in other ways that may consequently benefit the animals, eg by spending more money on quality feed.
The decrease in cost to produce the vaccine would allow the money spent on researching other diseases that could then benefit human and animal health.
The techniques and methodologies used in this experiment could be applied in the future to many other parasites and also to human health so the project is beneficial to all fields of medical research.
(b) Why do you wish to apply for a Vacation Scholarship and what are your career intentions at present? (no more than 100 words)
I have applied for the Vacation Scholarships because I hope to go into research in the future. I am fascinated by the science that underpins the practical side of veterinary medicine and hope to contribute to the wealth of knowledge our profession has in the future. I am particularly interested in immunology and the development of vaccinations as it can make a huge difference to a large number after they have been put to market. I would like to build on my current scientific skills and this project has many useful techniques which I would like to learn.
(c) Have you had any other research experience (apart from your course projects)? If yes, please describe. (no more than 100 words)
I have had little experience of research in the past which is another reason that I would like to do the Vacation Scholarship. In my last year of school I did a research project in my chemistry class on how much iron was in an iron tablet. This was self directed and I thoroughly enjoyed the experience as it taught me valuable scientific methods such as titration, correct weighing and avoiding contamination. It also taught me how to write up my experiments.
Part 2 : Critical evaluation of the proposed research project
9. PROJECT SUMMARY
Summarise this project in layman’s language (so that it is understandable by non-scientists) (no more than 200 words) [4 marks]
Lungworm in cattle is a disease caused by a roundworm called Dictycocaulus Viviparus. This worm infects the lungs causing respiratory disease and in some cases death. There is a vaccine for the disease th at uses larvae (young worms) that have been treated with radiation. This weakens the larvaes ability to cause disease and instead, these larvae cause the body to producing defence mechanisms against the worm so that in the future the animal cannot be infected. This is an effective vaccine but infected animals must be used to produce the larvae which is expensive and ethically questionable.
In this project we are researching the difference between larvae that have been treated with radiation and larvae that have not. By looking at their genetics and identifying the differences we can find the genes of the treated larvae that cause the body to produce immunity (defence mechanisms). With this
understanding of the current vaccine and the genes that allow it to work, there is the future possibility that a vaccine could be used that is made of protein and animals will not need to be used.
10. PROJECT STRENGTHS AND WEAKNESSES
What do you think are the strengths and weaknesses of this project ? (no more than 400 words – you should mention 2 strengths and 2 weaknesses, explaining your rationale for each ) [8 marks]
The strengths of the project are that more than one technique i s used to confirm the differential
expression of genes, both RNA sequence data and PCR. This will eliminate error and give more reliability to the results. By using bioinformatics tools such as the NCBI database, information is taken from a reliable source that has been tested and experimental error is removed from the results.
The weaknesses in this experiment are that live specimens are used which will have variation. The individual larvae may have differences in gene expression which may cause confession in identifying the genes that are causing Parasitic Bronchitis.
Another weakness is that these worms are developed in vitro which may be different from their development to L3 in the field due to environmental differences. Pathology is seen after day 7 in the body of the cow and the parasites continue to develop until day 26, which is when the clinical signs worsen. In this time the infective L3 change to L4 and then into adults so there may be changes in gene
11 – UNDERLYING SCIENCE
Describe treatments other than vaccination currently available for helminths of livestock and any problems associated with these treatments.
[you should refer to core course material delivered in BVMS1 and BVMS2 and other resources as appropriate] (no more than 400 words) [7 marks]
Faciola hepatica can be treated with anthelmintics. Acute infections will be treated with triclabendazole and subacute with closantel or nitroxymil. Triclabendazole is a member of the Benzimadaol group of anthelmintics which work by binding the tubilun of the helminths and starving them. The tubulin will be absent after 6-24 hours.
Ostergia is a nematode that causes disease in cattle. This can also be treated with anthelmintics, type 1 will be treated with the most currently available anthelmintics and type 2 will be treated with bezimadazoles or microcyclic lactones. The microcyclic lactones cause flaccid paralysis in the helminth by interfering with its neurotransmitters.
The above drugs are very effective on the helminths they target and have few side affects. However due to over use and incorrect use, resistance to the anthelmintics is a problem. (Nematodes of the GI tract, Tony Page, mod 4, week 2)
(Anthelmintics, Fiona Dowel, mod 4, week 3)
(Trematodes of veterinary significance, William Weir, mod 4, week 2)
Anthelmintic drugs also work in expelling lung worm. They can be used early to reduce the pathology that lungworm can cause but they are problematic because they kill the lungworm before an adequate immunity has been built up by the cow to prevent future lungworm infection. This means that while cows may survive their first season of infection they lack immunity the next year and may become severely infected. In animals are mildly infected with lungworm it is suggested that they are treated with anthe lmintics early and moved to clean pasture. In severely affected animals they should be housed so that they cannot be further infected and be given NSAIDs and antibiotics if pyretic.
12. PUBLISHED WORK
Provide 2 original references of relevance to the project and 1 review paper . State the relevance of the 2 original papers to the proposed research project (no more than 250 words) [5 marks]
Review Paper : (CANTACESSI, C., GASSER, R. B., STRUBE, C., SCHNIEDER, T., JEX, A. R., HALL, R. S., CAMPBELL, B. E., YOUNG, N. D., RANGANATHAN, S., STERNBERG, P. W. & MITREVA, M. 2011. Deep insights into Dictyocaulus viviparus transcriptomes provides unique prospects for new drug targets and disease intervention. Biotechnol Adv, 29, 261-71.)
Origional papers:
Paper 1: (STRUBE, C., BUSCHBAUM, S. & SCHNIEDER, T. 2012. Genes of the bovine lungworm Dictyocaulus viviparus associated with transition from pasture to parasitism. Infect Genet Evol, 12, 1178-88.)
This paper is relevant to the project as it is also studying the genes of the Dictyocaulus Viviparus at the L3 infective stage. The genes found as a result of this paper could give support to our own findings. By
separating the genome into ESTs and using 'conceptual methods' (which could include the RNA sequencing and bioinformatics that our project is using) the pathogenesis-related proteins (PRP) have been found. This matches the aim of our project which is to find the pathogenic proteins of the L3 that are causi ng immune response.
The research goes further in looking at up and down regulations of the parasite genes as they develop past the L3 stage. After our own project has identified pathogenic proteins this would allow us to define which of these would still be important after the parasite has entered the body of the host, narrowing down our results. Furthermore, the data found can act as a basis for vaccine development which is also the aim of the research project .
Paper 2 : (MANGIOLA, S., YOUNG, N. D., STERNBERG, P. W., STRUBE, C., KORHONEN, P. K., MITREVA, M., SCHEERLINCK, J. P., HOFMANN, A., JEX, A. R. & GASSER, R. B. 2014. Analysis of the transcriptome of adult Dictyocaulus filaria and comparison with Dictyocaulus viviparus, with a focus on molecules involved in host-parasite interactions. Int J Parasitol, 44, 251-61.)
This paper is relevant to the research project because they both have similar aims. This paper identifies nematode to host interactions and our own project aims to identify the proteins that cause immune response by interacting with the host. Both use comparative transcriptomic analysis with an aim to improve the understanding of Dictyocaulus Viviparus. Although this research is comparing the genes of different species of D. Dictyocaulus, the methods could also be applied to comparing irradiated and non-irradiated larvae and RNA-seq is used in both experiments. The role of type 2-like crytatin, which is a protein, is characterised by looking at their role in many different nematodes including lungworm. This research is linked to the project as the second step is to find the function of differently expressed genes .
13. NEXT STEPS
If the aims of this project were successfully achieved, what would you suggest would be a follow on investigation to progress knowledge in this area ? (no more than 150 words) [3 marks]
From the results of the project we have a better understanding of
how the larval vaccine works and the genes present in the irradiated larval vaccine that produce proteins that cause the immune response. The next step in confirming the function of these proteins would be to clone the genes and test them on a host antigen presenting cell to see if an immune response occurs. From here a vaccine could be developed using these proteins which would then be trial tested.
14. INSTITUTIONAL CONTEXT
How does this project relate to the research interests of the University of Glasgow ? [ you should refer to the University web site information on research in the Institutes and Schools
http://www.gla.ac.uk/colleges/mvls/ ] (no more than 150 words) [3 marks]
One of the Institute of Infection Immunity and Inflammations aims is to study parasitic pathogens in animals and this project falls under this heading.
Parasitology research at the University aims to 'improve the control and treatment of parasitic disease' (Glasgow University, Institute of Infection, Immunity and Inflammations, Parasitology) which is also the aim of this project.
There has been a lot of research on the topic of lungworm at Glasgow University and the Huskvak vaccine was developed at Glasgow vet school. As this project is the next step in the understanding and development of treating lungworm, it is very much in the Universities interests.
