Animal & Veterinary Sciences

After completing a PhD (Biochemistry) at Monash University in 1977, I undertook postdoctoral studies at Mahidol University (Thailand), Colorado State University and UCLA (1977-1981). Since 1981, I have held positions as a Research Scientist at Walter and Eliza Hall Institute, Head of Department at Victorian Institute of Animal Science, Associate Professor in Biochemistry and Molecular Biology at Monash before joining McGill University (Canada) in 2001 as Director of the Institute of Parasitology where I held a prestigious Canada Research Chair in Immunoparasitology (2002-7). In 2007, I was appointed Strategic Research Professor in the School of Animal and Veterinary Sciences at CSU, where I will continue my work discovering molecular knowledge of parasite functions in order to develop vaccines or drugs to control infection. My specific research interests include the molecular biology and protein biochemistry of eukaryote parasites and the development of diagnostic tools using biomarker approaches.

 

top of page ^

Current PhD and postdoc projects in the lab

I have several PhD projects available for study that offer the opportunity to perform cutting edge research using the techniques of immunology, molecular biology and protein chemistry. Students who would be competitive to obtain student scholarships, have an interest in studying medical or veterinary diseases and would like to acquire training in modern molecular techniques in a stimulating environment are encouraged to contact me by email tspithill@csu.edu.au or tel 02-6933 2439.

            We are studying the host and parasite factors underpinning immunity to fasciolosis using several approaches:

1. using proteomics to analyse host plasma and macrophage biomarkers associated with expression of immunity within 6 weeks of infection in cattle;
2. using monocyte/ macrophages and sera from cattle vaccinated by a drug-abbreviated infection to define the cells, IgG isotype and effector molecules in cattle that are involved in ADCC killing of F. hepatica NEJ in vitro.
3.  using proteomics to analyse surface-exposed juvenile parasite proteins that are potential targets of this immunity.

APAI Award available immediately in my lab

PhD studies in population genetics of triclabendazole resistance in liver fluke parasites

The ARC has recently funded the Linkage Project “Liver fluke: improving disease control through understanding of parasite diversity, drug resistance and better diagnosis”  by Profs Terry Spithill, Nick Sangster and Drs Tiggy Grillo at CSU and Glenn Anderson at Virbac Australia Pty Ltd.

This project aims to improve our ability to control liver fluke infection in livestock through a better understanding of the underlying basis of treatment failure and the development of improved diagnostic tools to promote better use of antiparasitic drugs. The major aims of the project are to:

• define the existing genetic diversity and population structure of F. hepatica including populations susceptible and resistant to the drug triclabendazole;
• develop superior diagnostic tests for liver fluke infection;
•  apply these diagnostic improvements to measure the extent of resistance in the field in NSW and N Victoria. This information will inform management decisions on parasite control.

The APAI student will:

• use genetic markers and karyotyping to examine wild-type fluke populations from sheep and cattle in NSW and N Victoria to define the level of diversity and chromosomal ploidy in a single host animal, and within host populations.
• examine the inter population diversity between F. hepatica isolated from cattle and sheep, and between populations collected from diverse geographical locations
• compare this diversity with flukes from animals not responding to triclabendazole treatment to determine whether “resistant” flukes exhibit lower diversity and distinct haplotypes and genotypes.

This project provides a sound basis for PhD training in molecular genetics, molecular phylogeny and parasite biology.

The stipend is $26,140 pa for 3 years plus a relocation allowance of up to $6000.

For details, contact Terry Spithill (tspithill@csu.edu.au; 02-6933 2439) or Nick Sangster (nsangster@csu.edu.au; 02 69334107).

The work is based at the Wagga Wagga campus in new PC2 facilities at the School of Animal and Veterinary Sciences
(see http://www.csu.edu.au/faculty/science/savs/ ).

Project 1: Identify biomarkers associated with expression of acquired immunity in cattle.
            Cattle express acquired resistance early after drug-abbreviated infections and killing of flukes occurs within 6 weeks of challenge during the parenchymal phase of infection. We will focus on the time window from 1-6 weeks after infection and carefully examine biomarkers in plasma and activated monocyte/ macrophages (recovered from donor cattle) of treated vs non treated cattle. This study will document for the first time the induction of key plasma/macrophage proteins associated with acquired immunity against F. hepatica in cattle and allow us to form new hypotheses about effector pathways operating in cattle.

Project 2: Linking in vivo and in vitro approaches by defining bovine immune effector mechanisms that kill F. hepatica in vitro

We will use monocyte/ macrophages and sera from cattle vaccinated by a drug-abbreviated infection to define the cells, IgG isotype and effector molecules in cattle that are involved in ADCC killing of F. hepatica NEJ using our in vitro assay system. The effector molecule(s) active in cattle is unknown but will be defined using specific inhibitors in the killing assay (eg. SOD, NMMA) to assess the relative levels of ROS and NO produced under our assay conditions. Monocyte/macrophages will be incubated with/without the different antibody fractions and killing of NEJ of F. hepatica will be estimated.

Project 3: Identify and characterize surface-exposed tegument proteins expressed in F hepatica and recognised by sera from vaccinated cattle
Postdoctoral fellow: Dr Janelle Wright
            We are starting to define tegument proteins expressed on adult parasites using several biochemical approaches. We will use the tegument biotinylation technique to purify and identify tegument proteins on adult and juvenile parasites using proteomic approaches. We will first establish the technique with adult flukes and then explore the juvenile tegument. Surface-biotinylated proteins will be enriched using streptavidin, the proteins separated by one or 2D gel electrophoresis, protein bands/spots eluted and digested with trypsin. Protein identities will be obtained either by peptide mass fingerprinting and matching to existing EST databases for F. hepatica or by direct MS/MS analysis to generate amino acid sequences.
            Only a subset of these proteins will be targets for ADCC against F hepatica. We will perform western blot analyses to determine which of the surface tegument proteins, enriched by streptavidin binding, are also targets of IgG1 or IgG2 responses in immune cattle. By focussing only on those proteins that are selectively enriched by biotinylation-streptavidin isolation, we will determine those proteins that are surface exposed and antigenic in cattle during the time window (weeks 1-6 post challenge) when acquired immunity is expressed.

Honours and summer student projects for 2008-9

I am seeking highly motivated students interested in applying molecular approaches to study parasites and infectious organisms. Training is offered in the techniques of proteomics, cell and molecular biology and tissue culture. Note that the School has several Honours and Summer scholarships available for highly ranked applicants.

Honours Project #1
Surface tegument development of Fasciola hepatica under different culture conditions in vitro
with Prof Spithill and Dr Janelle Wright
At present, there is no commercial vaccine for controlling fasciolosis; we are interested in developing a vaccine by targeting surface proteins expressed on the surface tegument of the parasite since we know that immunity to Fasciola in sheep involves an antibody-mediated killing mechanism directed to these surface tegument proteins. However, the parasite is able to evade the host’s immune system by varying the tegumental proteins as it develops from a juvenile to adult parasite within the host but the surface proteins expressed by the parasite have  not yet been well defined.

1. The first aim of this project is to culture Fasciola hepatica parasites from juvenile stages through to young adults in the presence of various media to determine the effects they have on the growth and development of the parasite.
2. The second aim is to examine, using proteomics and microscopy techniques, tegument changes during the development process.  The student will receive training in tissue culture, proteomics, cell biology, bioinformatics and Parasitology.

Honours Project #2
Identification of Staphylococcus aureus biomarkers in milk of Dairy Cows
With Prof Spithill, Dr Jan Lievaart and Dr Janelle Wright.
Milk from cows with mastitis (S. aureus infections) is unsafe for human consumption and detection of an infection is critical to food safety. At present, bacterial culture is performed on milk products but these tests can takes days  and often results are returned as negative or inconclusive. These poor results may be due to irregular distribution of S. aureus in the cow’s udder or contamination of other bacteria. For successful treatment of S. aureus, infected cows need to be diagnosed quickly, and treated to minimise spread of the pathogen to the herd.

1. The first aim of this project is to screen milk samples from control and infected cows for somatic cell count irregularities.
2. The second aim is to compare, using 1D and 2D electrophoretic techniques, protein profiles of each of these samples. Novel protein markers observed in infected milk will be characterised using MALDI-TOF MS/MS and bioinformatics. With time permitting, antisera will be generated to biomarkers of interest and used for ELISA and western blot analysis to potentially diagnose mastitis. The student will receive training in microbiology, proteomics, cell biology and bioinformatics.

Current trainees in my lab

Marie-Claire Rioux, Institute of Parasitology, McGill University. PhD Thesis: “Biomarkers for Fasciola infection”.

Janelle Wright, Postdoctoral fellow, CSU: “Proteomics of the Fasciola tegument”.

Michelle Ayton, Research Assistant

 

top of page ^

Research Interests

My recent research has focused on the parasites Fasciola (liver fluke) and Plasmodium (malaria) studying areas such as host immune responses to parasites, parasite immune evasion mechanisms, parasite proteomics and vaccine and drug discovery. We are characterising parasite molecules that determine virulence and pathogenesis using genomic/proteomic approaches with the long term aim of developing vaccines based on these molecules.

I also have an interest in biomarkers of disease and developing new diagnostics based on these biomarkers. One new project in my lab aims to look at biomarkers of mastitis in milk (see below). We are also interested in evaluating new drugs as potential chemotherapies for F. hepatica. We recently showed that DNA binding drugs are highly effective anti-malarials ( see Yanow et al, 2008) and we are interested to determine if such drugs are active against other parasites.

I am looking for highly motivated graduate students and postdoctoral fellows to tackle the challenges caused by these diseases. My research has a deliberate international outlook with current collaborations in Sudan, Thailand, China, India, UK, Canada and Australia.

Liver fluke disease and vaccine development

Liver fluke infection (fasciolosis) is a major neglected zoonotic disease caused by Fasciola hepatica and F. gigantica, flatworm parasites transmitted following ingestion of infective metacercariae encysted on aquatic plants. Liver flukes are a serious constraint on global livestock production, with  >600m animals at risk of fasciolosis and economic losses of >US$3b p.a. (see Piedrafita et al, 2004).  In Australia, economic losses are $60-90m p.a, with tens of millions of sheep/cattle exposed to the infection. Fasciola is also a significant food borne zoonosis recognised by the WHO and human cases of fasciolosis have been reported in Australia including work from my laboratory (Hughes et al 2003). There is no commercial vaccine for this disease. My lab is examining acquired immune mechanisms expressed by sheep and cattle that are involved in killing juvenile liver flukes with a view to vaccine development.

Current experimental fluke vaccines, including work from my lab, can only achieve partial protection, manifested by reductions in fluke burdens of 38-72% in cattle. Although some vaccinates show >90% reduction in fluke burdens, a major constraint for vaccine design is the fact that we do not know which effector mechanisms in cattle actually control infections (ie kill the parasite) or why some animals respond better than others. This hinders our ability to lift vaccine efficacy to commercially desirable levels.

Multiple published studies provide strong evidence that cattle can acquire high levels of resistance (80-98%) to Fasciola following vaccination using irradiated metacercariae, drug abbreviated infection, parasite extracts or defined antigens (reviewed in Piedrafita et al 2004). These observations show that Fasciola antigens can induce protective immune responses in cattle and that a defined commercial vaccine that achieves >80% protection is a theoretically achievable goal if we can better understand how the immune system controls fluke infections.

Data from different studies in cattle and sheep showed that immune killing of parasites occurs within 6 weeks of infection but only after some damage occurs to the liver parenchyma, suggesting that the newly excysted or immature juvenile (NEJ) parasite, not the adult parasite, is the primary target of the effective immune response in the liver.  However, we do not know which effector mechanisms kill liver flukes within 6 weeks of infection or the parasite antigens that are the target of this response.

We were the first to show that Fasciola NEJ are susceptible to antibody-dependent cell cytotoxicity (ADCC) mediated by superoxide radicals produced by sheep macrophages in vitro (Piedrafita et al 2007). Our results suggest two important conclusions;

1. that macrophages, acting in concert with specific antibody, are critical effector cells involved in the resistance to liver fluke but we need to know more about how the host activates macrophages and kill flukes
2. that antigens on the surface of juvenile/immature flukes, recognised by immune sera, are the targets of the ADCC mechanism and we need to know more about these antigens.

 

top of page ^

Recent publications last 5 years

A complete list of my publications can be found on the PubMed web site at http://www4.ncbi.nlm.nih.gov/entrez/query.fcgi

• Irving, J., SPITHILL,T.W., Pike, R.N., Whisstock, J.C. and P. M. Smooker. The evolution of enzyme specificity in Fasciola spp. J Mol Evolution  57:1-15 (2003).
• Hughes, A.J., SPITHILL, T.W., Smith , R.E., Boutlis, C. and Johnson, P.D.R. Human fasciolosis acquired in an Australian urban setting. Med J Aust 178:244-5 (2003).
• Rainczuk, A, Smooker, P.M, Kedzierski, L., Black, C.G, Coppel, R.L, and SPITHILL, T.W.  The protective efficacy of MSP4/5 against lethal P.chabaudi adami challenge is dependent on the type of DNA vaccine vector type and vaccination protocol. Vaccine 21: 3030-42. (2003).
• Rainczuk, A., Scorza, T., Smooker, P.M, and SPITHILL, T.W. Induction of Specific T-cell responses, opsonizing antibodies, and protection against Plasmodium chabaudi adami infection in mice vaccinated with genomic expression libraries expressed in targeted and secretory DNA vectors. Inf Immun 71:4506-4515 (2003).
• Boag P.R., Parsons, J.C., Presidente, P..J, SPITHILL, T.W. and Sexton, J,L. Characterisation of humoral immune responses in dogs vaccinated with irradiated Ancylostoma caninum. Vet Immunol Immunopathol. 92: 87-94 (2003).
• Law, R.H.P., Smooker, P.M., Irving, J.A., Piedrafita, D., Ponting, R., Kennedy, N.J., Whisstock, , J.C., Pike, R.N. and SPITHILL, T.W. Cloning and expression of the major secreted cathepsin B-like protein from juvenile Fasciola hepatica and analysis of  immunogenicity following liver fluke infection. Inf Immun 71:6921-32 (2003).
• Piedrafita, D., Raadsma, H., Prowse, R. and SPITHILL, T.W. Immunology of the Host Parasite Relationship in Fasciolosis (Fasciola hepatica and Fasciola gigantica). Invited Review. Can J Zoology 82: 233-250 (2004).
• Smooker, P.M., Rainczuk, A., Kennedy, N. and SPITHILL, T.W. DNA vaccines and their application against parasites: promise, limitations and potential solutions. Biotechnology Annual Reviews 10:  189-236  (2004).
• Rainczuk, A., Scorza, T., SPITHILL, T.W. and Smooker, P.M. A bicistronic DNA vaccine containing Apical Membrane Antigen-1 and Merozoite Surface Protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent P. chabaudi adami DS malaria. Inf Immun 72: 5565-5573 (2004).
• Scorza, T., Grubb, K.,  Smooker,P., Rainczuk , A., Proll, D. and SPITHILL, T.W.  Induction of strain-transcending immunity against Plasmodium chabaudi adami malaria with a multiepitope DNA vaccine. Inf Immun 73: 2974-2985 (2005).
• Kennedy, N.J., SPITHILL, T.W., Tennent, J., Wood, P.R. and D. Piedrafita. DNA vaccines in sheep: CTLA-4 targeting and CpG motifs enhance immunogenicity in a DNA prime/protein boost strategy. Vaccine 24: 970-979 (2006).
• SPITHILL, T.W. Crystal Ball issue: Engineering transgenic P falciparum to propagate in mice. Parasite Immunol. 28: 235-269 (2006).
• Yanow, S.K., Purcell, L.A. and SPITHILL, T.W. The A/T-specific DNA alkylating agent Adozelesin inhibits P. falciparum growth in vitro and protects mice against P. chabaudi  infection. Mol Biochem Parasitol. 148: 52-59 (2006).
• Beckham, S.A., Law, R.H., Smooker,P.M., Quinsey, N.S., Caffrey, C.R., McKerrow, J.H., Pike, R.N. and  T.W. SPITHILL. Production and processing of a recombinant Fasciola hepatica cathepsin B-like enzyme (FhcatB1) reveals potential processing mechanisms in the parasite. Biol. Chem  38: 1053-1061. (2006)
• Boscariol, R., Pleasance, J., Piedrafita, D.M., Raadsma, H.W. and SPITHILL, T.W. Sequence of cDNAs for two allelic forms of ovine CD4 predicts a Ser183/Pro183  polymorphism in the coding sequence of domain 3. Vet Immunol Immunopathol 113: 305-312 ( 2006)
• Raadsma,  H.W., Kingsford, N.M., Suharyanta, SPITHILL, T.W. and Piedrafita. D. Host responses during experimental infection with Fasciola gigantica or Fasciola hepatica in Merino sheep I. Comparative immunological and plasma biochemical changes during early infection. Vet Parasitol. 143: 275-286 (2007).
• Piedrafita,D.M., Estuningsih, E., Pleasance, J., Prowse, R.K., Raadsma,H.W., Meeusen, E.N.T. and T.W. SPITHILL. Peritoneal lavage cells of Indonesian thin tail sheep mediate antibody-dependent superoxide radical cytotoxicity in vitro against newly excysted juvenile Fasciola gigantica but not juvenile Fasciola hepatica. Inf. Immunity 75:1954-1963. (2007).
• Yanow, S.K., Purcell, L.A., Lee, M., Spithill, T.W. Genomics-based drug design targets the AT-rich malaria parasite: implications for anti-parasite chemotherapy. Pharmacogenomics  8: 1267-1272 (2007).
• Rioux, M.C., Carmona, C., Acosta, D., Ward, B., Ndao, M. and T.W.  SPITHILL. Discovery and validation of serum biomarkers over the first twelve weeks of Fasciola hepatica infection in sheep. (invited paper) Int J Parasitol. 38:123-36 (2008)
• Yanow, S.K*., Purcell, L.A*., Pradel, G., Sato, A., Rodriguez, A., Lee, M., Spithill, T.W. Potent antimalarial and transmission-blocking activities of a novel DNA binding agent. J Inf Diseases 197:527–534 (2008).* joint first author.
• Scorza, T., Grubb, K., Cambos,M., Santamaria, C., Tshikudi Malu, D. and T. W. SPITHILL. Vaccination with a Plasmodium chabaudi adami multivalent DNA vaccine cross protects A/J mice against challenge with P c adami DK and virulent P. c. chabaudi AS parasites. Int J Parasitol 38: 819-827 ( 2008).
• McGonigle, L., Mousley, A., Marks, N.J., Brennan, G.P., Dalton, J.P., SPITHILL, T.W., Maule, A.G. The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference. Int J Parasitol  38:149-155 (2008).
• Smith, R.E., SPITHILL, T.W., Pike, R.N., Meeusen, E.N.T., D. Piedrafita. Fasciola hepatica and Fasciola gigantica:  Cloning and characterisation of 70kDa heat-shock proteins reveals variation in HSP70 gene expression between parasite species recovered from sheep. Exp Parasitology 118:536-542 ( 2008).
• Purcell, L.A., Yanow, S.K., Lee, M., SPITHILL,T.W. and Rodriguez, A. Chemical attenuation of Plasmodium berghei sporozoites induces sterile immunity in mice. Inf Immunity 76:1193-9 (2008).
• Raadsma H. W., Kingsford N.M, Suharyanta, SPITHILL, T.W and Piedrafita D. Host responses during experimental infection with Fasciola gigantica and Fasciola hepatica in Merino sheep: II Development of a predictive index for Fasciola gigantica worm burden. Vet Parasitology 154: 250-61 (2008).
• Purcell, L.A., Wong, K.A., Yanow, S.K., Lee,M., SPITHILL, T.W. and A. Rodriguez. Chemically attenuated Plasmodium sporozoites induce specific immune responses, sterile immunity and cross-protection against heterologous challenge. Vaccine  26:4880-4884 (2008).
• Estuningsih, S.E., SPITHILL, T.W., Raadsma H.W., Law, R.H.P.,  Adiwinata, G., Meeusen, E.,  and Piedrafita, D. Development and application of a faecal diagnostic sandwich ELISA for estimating prevalence of Fasciola gigantica in cattle in central Java, Indonesia. J. Parasitology (in press 2008).

 

top of page ^

Recent graduates from my lab - Where are they know?

2007.  Lisa Purcell, Institute of Parasitology, McGill. PhD Thesis: “Genetic and biochemical strategies to block the transmission cycle of the malaria parasite”. Now a postdoctoral fellow at Regeneron, New York. http://www.regeneron.com/

2006. Rebecca Smith, Department of Biochemistry and Molecular Biology, Monash University. PhD Thesis: " Cloning and characterisation of pathogenesis factors of Fasciola hepatica using cDNA representational difference analysis and signal sequence trap approaches” . Now a postdoctoral fellow at Univ. Melbourne.  

2005. Simone Beckham, Department of Biochemistry and Molecular Biology, Monash University. PhD Thesis: “ Characterisation of cathepsin proteases from Fasciola hepatica”. Now a postdoctoral fellow at Monash.

2005. Nick Kennedy, Department of Biochemistry and Molecular Biology, Monash University. PhD Thesis: “DNA vaccines for Fasciola hepatica”. Now a postodoctoral fellow at Monash.

2005: Kim Grubb, Institute of Parasitology, McGill. MSc Thesis: “Malaria vaccine antigens”. Now in the business sector in Montreal.

2005: Cynthia Santamaria, Institute of Parasitology, McGill. MSc Thesis: “Merozoite surface proteins in malaria”. Now in research assistant at the McGill University Health Centre, Montreal.

2004. Adam Rainczuk, Department of Biochemistry and Molecular Biology, Monash University. PhD Thesis: “Evaluation of expression library immunisation as a tool for malaria vaccine discovery”.  Now Witchery Research Fellow at Prince Henry's Institute for Medical Research, Melbourne.

2004: Rya Boscariol, Institute of Parasitology, McGill. MSc Thesis: “Functional characterisation of Sheep CD4 and susceptibility to cleavage by Fasciola proteases”. Now studying medicine at Queen’s University, Ontario.

 

 

top of page ^