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Innate Resistance Dichotomy to Bacterial Infections in Different Breeds of Cattle

Principal supervisor: Professor Dirk Werling / Dr Amanda Gibson (RVC)

Co-Supervisor: Professor Brendan Wren / Dr Sam Willcocks (LSHTM)

Cows are susceptible to infection from pathogenic organisms commonly present in the farm environment. Control of infection is difficult; it can only partially be controlled with antibiotics, vaccine and biosecurity measures. Novel approaches to disease prevention need to be established and one approach includes breeding for disease resistance to a wide range of pathogens by selecting for the “fittest” innate immune system. Recent evidence suggests that some cattle breeds are more resistant to bacterial infections. Indeed, our own data have shown that there are differences in the ability of macrophages (MØ) generated from Brown Swiss and Holstein-Friesean cattle to harbour and kill Salmonella enterica serovar Typhimurium (S.typhimurium). To our knowledge, this is the first time that differences of this magnitude in response to intracellular bacteria have been observed in cattle.

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), and Listeria (L.) monocytogenes, the causative agent of listeriosis, are intracellular pathogens capable of invading and living within MØ. Both diseases have zoonotic potential, being involved in either tuberculosis or human listeriosis. Listeriosis affects primarily pregnant women, newborns, adults with weakened immune systems, and the elderly, with a mortality rate of about 20%. Inside the MØ these bacteria are capable of surviving and dividing whilst out of the reach of the adaptive immune response. Resistance to intracellular pathogens has been shown to have an element of genetic determination. In mice, genetic polymorphism of SLC11A1 (formerly Nramp1) has been shown to be associated with resistance to infection with Salmonella, Mycobacteria and Listeria (1). In humans, genetic association studies have also shown a degree of association between polymorphic variants of SLC11A1 and resistance to Leishmania (2). However, although SLC11A1 has been identified in cattle, no association has been shown between polymorphism and resistance to infection with intracellular pathogens. Nramp1 is expressed in the phagosomal membrane and presumably mediates bacterial killing by sequestering iron uptake. Subsequently, autophagy is responsible for bacterial killing by promoting fusion of, for example, mycobacterial phagosomes to lysosomes. At the same time, activation of autophagy blocks activation of the inflammasome complex NLPR3, which is further inhibited by the production of nitric oxide (NO). Both expression of NO as well as inhibition of inflammasome activation result in a reduced pro-inflammatory response as well as reduced expression of anti-microbial peptides (AMP), and impact also on the expression of the purinergic receptor P2X7, which is involved in anti-mycabcterial activity (3). As genetic polymorphisms were identified for some molecules involved in autophagy/NLRP3 activation in the innate immune response to Salmonella between the two breeds, we now wish to extend the experiment using state-of-the-art techniques and assess differences in MØ activation on a larger scale. Here, high-throughput sequencing of RNA (RNASeq) will help to primarily analyze global gene-expression in MØ from both breeds. In addition, RNASeq is also an efficient way to discover coding SNPs. The approach proposed for this work can potentially provide the basis for development of simple assays linking genotype with phenotype. Our hypothesis is that genetic selection to produce the modern high yielding dairy cow has inadvertently been associated with a reduced ability to mount an adequate innate immune response. This makes animals potentially less able to resolve common infections requiring defence at mucosal surfaces. Our aim is to identify key genes in affected signalling pathways involving innate immune function, which could subsequently impact on breeding strategies using marker assisted selection to counteract their reduced innate immune response.

Candidate requirements

The student will attend the UCL led doctoral training partnership course within the first year with mini-projects contributing to the PhD. This will involve training in analysing RNASeq data, as well as potentially generating their own data-set. Subsequently, the proposed project will utilize cutting-edge technologies that RVC and LSHTM have invested in over recent years: analysis of gene expression profile by RNASeq, verification of differences by multiplex ELISA and qPCR, as well as sequencing of candidate genes and polymophism analysis. In addition, the project will build upon the LSHTM experience using techniques established in the context of M.bovis research. The project will establish a novel approach to studying the key events following L.monocytogenes and M. bovis infection of its primary target cells the Mφ. These cells, which can readily be established from peripheral blood would lessen the need to conduct infection of animals, with significant ethical and economic benefits. The project should provide important information on the interactions of two different bacteria causing diseases of economic importance with Mφ which could form the basis for further funding proposals seeking to exploit this for the development of improved vaccines or therapeutics. A BSc/MSc in Immunology with experience of qPCR, sequencing and cell culture techniques would be great advantage for this project.

Key references

1.) Araujo, L. M., O. G. Ribeiro, M. Siqueira, M. De Franco, N. Starobinas, S. Massa, W. H. Cabrera, D. Mouton, M. Seman, and O. M. Ibanez. 1998. Innate resistance to infection by intracellular bacterial pathogens differs in mice selected for maximal or minimal acute inflammatory response. European journal of immunology 28: 2913-2920.

2.) Mohamed, H. S., M. E. Ibrahim, E. N. Miller, J. K. White, H. J. Cordell, J. M. Howson, C. S. Peacock, E. A. Khalil, A. M. El Hassan, and J. M. Blackwell. 2004. SLC11A1 (formerly NRAMP1) and susceptibility to visceral leishmaniasis in The Sudan. European journal of human genetics : EJHG 12: 66-74.

3.) Wewers, M. D., and A. Sarkar. 2009. P2X(7) receptor and macrophage function. Purinergic signalling 5: 189-195.

4.) Cho, J., and D. G. Lee. 2011. The antimicrobial peptide arenicin-1 promotes generation of reactive oxygen species and induction of apoptosis. Biochimica et biophysica acta 1810: 1246-1251.

Further details about the project may be obtained from:

Principal Supervisor: Professor Dirk Werling, dwerling@rvc.ac.uk

Co-Supervisor: Professor Brendan Wren, brendan.wren@lshtm.ac.uk

Further information about PhDs at Royal Veterinary College is available from:

http://www.rvc.ac.uk/research

Application forms and details about how to apply are available from:

http://www.rvc.ac.uk/study/postgraduate/phd/how-to-apply

Mrs Carole Tilsley
Research Degrees Admissions Officer
Graduate School
Royal Veterinary College
Email:  researchdegrees@rvc.ac.uk
Tel No:  44 (0) 20 7468 5134

There are a number of documents which you will need to upload to your UKPass application form:

  • CV
  • A personal statement explaining why you would like to undertake the project you are applying for
  • An electronic copy of your degree certificate(s)
  • A transcript of your degree(s) - this must be a certified translation if the original was not issued in English
  • 2 confidential references. Please note that it is your responsibility to contact your referees and arrange for references to be provided. These should be on letter headed paper, contain the signature of the referee and should be scanned and uploaded with your UKPASS application these can also be submitted separately to researchdegrees@rvc.ac.uk. Further information is available on our website.

Closing date for applications is 30 March 2015