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Plant Antibody Delivery of Passive Immunisation against E.coli 0157:H7: A Novel Means of Control in the Animal. - OZ0703

Description
Escherichia coli 0157 is recognised as the causal agent of a number of severe human diseases. Recently, evidence has been gained that the ovine is also a potential reservoir. The bacteria can be excreted in faeces and found in animal manure or slurry which can, in turn, be the source of environmental and water contamination. There is also evidence that the bacterium is spread between animal carcasses through contamination by faecal matter during slaughter process.

Current MAFF-funded research is investigating colonisation, persistence and epidemiology of E. coli 0157:H7 in cattle. Existing means of protecting the food chain are directed towards improving food hygiene. Clearly, it is also important of work towards improved and novel methods of control in the animal so that the infectious challenge to food hygiene controls is reduced.

A key aim of this project is to express recombinant antibodies in edible crop plants, to facilitate the delivery of passive immunisation against Escherichia coli 0157:H7 to mucosal surfaces of livestock. This method of production and delivery of passive immunisation will have universal applications for sustainable livestock production systems. All of the individual components of the project have been achieved, which are, raising specific antibodies against target antigens, construction of recombinant antibodies, plant transformation, plant antibody expression and passive immunisation of mucosal surfaces. It is the intention to combine these technologies which represent not only a novel aspect of the project but also delivers a tangible demonstration of welfare benefit to the livestock industry.

The results of this research will provide considerable benefits. A dangerous and, currently poorly understood food hazard will be controlled. A particular advantage of the proposed control method is that, although providing a remedy to food contamination and a human health hazard, neither the food nor the human population is required to be treated.
Objective
01 Preparation of primary immune reagents against EHEC E.coli 0157:H7 CVL

02 Contruction of recombiant antibodies against 0157:H7 ADAS/Leics
a) monoclonal hybridoma cells will be used as a source of antibody gene sequences for recombinant antibody construction.
b) spleen cells from rabbit immunised with formalin fixed 0157:H7 will be used as a source of antibody gene sequences to construct a large library of phage antibodies for selection of binders to surface antigens.

03 Expression of antibody single chain variable fragment (scFv) genes in transgenic plants and confirmation of specificity ADAS/Leics
a) scFv with desired characteristics will be sub-cloned for plant expression
i) transient expression in tabacco leaves for rapid confirmation of expression in planta
ii) for larger-scale expression and to complete in vivo trials, selected scFv genes will be sub-cloned in expression vectors and used for stable expression in tabacco seed.
c) confirmation of plant deived antibodies' specificity CVL

04 in vitro and in vivo inhibition of attaching and effacing pathology CVL
a) 0157 adheres to an dmediates cytoskeletal changes, akin to the effeacement of gut epithial microvillious cells, in Hep-2 cells grown in vitro. The key mediators of this phenotype are Eae and TIR and plant antibodies against these will be tested for inhibition of this phenotype. Adherence will be assessed by standard protocols and inhibition of cytoskeletal changes monitored by FAS.
b) Individual chicks housed in category III isolaters will be fed known amounts of crushed tobacco seeds within the normal ration. Depending upon the specificity of the plant derived antibody, the presumed efficacy as deduced from in vitro tissue culture adherence assays and the likely yield of the antibody within the tabacco seed, the feeding regime will be aimed at determining the minimum amount required to inhibit the formation of attaching and effacing lesions in the duodenal loop of 14 day old chicks fed 100,0000 cfu EHEC 0157:H7.
05 Final report and recommendations for further research ALL
Project Documents
• Final Report : Plant antibody delivery of passive immunisation against E.coli 0157:H7: a novel means of control in the animal   (171k)
Time-Scale and Cost
From: 1999

To: 2003

Cost: £354,108
Contractor / Funded Organisations
Veterinary Laboratories Agency
Keywords
Animal Health              
Biotechnology              
Control              
E.coli O157              
GM Non-Food              
Plants and Animals              
Zoonoses              
Fields of Study
Animal Health