Improvements to the immunoassay for detection of Rathayibacter toxicus in hay
A. M. Masters A B , B. Samarasinghe A , M. J. Kalkhoven A , G. L. den Hollander A and D. G. Palmer AA Department of Agriculture and Food Western Australia, 3 Baron-Hay Court, South Perth, WA 6151, Australia.
B Corresponding author. Email: anne.masters@agric.wa.gov.au
Crop and Pasture Science 62(6) 523-530 https://doi.org/10.1071/CP10337
Submitted: 20 October 2010 Accepted: 24 May 2011 Published: 7 July 2011
Abstract
An improved protocol for the previously described enzyme-linked immunosorbent assay for Rathayibacter toxicus in hay is described. The improvements were driven mainly by the export hay industry requirement of same-day turnaround for testing of hay extracts. The preparation of hay extracts was shortened by 8 h. The time for adding samples to the enzyme-linked immunosorbent assay plates was shortened by the use of sample tubes with penetrable stoppers combined with specially designed racks. The monoclonal antibody used in the original protocol was purified and conjugated to horseradish peroxidase. This eliminated the need for a secondary step with an anti-mouse horseradish peroxidase conjugate and thereby shortened the assay by over 1 h. Results with the improved assay protocol showed a very high correlation with results obtained with the original protocol (r = 0.98). The assay is still sensitive enough to detect antigen equivalent to less than 1 average gall per kg of hay. These cost-effective changes have streamlined the testing of large numbers of samples for the presence of R. toxicus, in support of the hay export industry.
Additional keywords: ARGT, conjugation, corynetoxins, ELISA, IgM.
References
Bertozzi T, Davies KA (2009) Anguina paludicola sp. n. (Tylenchida: Anguinidae): the nematode associated with Rathayibacter toxicus infection in Polypogon monspeliensis and Lachnagrostis filiformis in Australia. Zootaxa 2060, 33–46.Bird AS, Stynes BA (1977) The morphology of a Corynebacterium sp., parasitic on annual rye grass. Phytopathology 67, 828–830.
| The morphology of a Corynebacterium sp., parasitic on annual rye grass.Crossref | GoogleScholarGoogle Scholar |
Edgar JA, Cockrum PA, Stewart PL, Anderton NA, Payne AL (1994) Identification of corynetoxins as the cause of poisoning associated with annual beardgrass [Polypogon monspeliensis (L.) Desf.] and blown grass (Agrostis avenacea C. Gemelin). In ‘Plant-associated toxins: agricultural, phytochemical and ecological aspects’. (Eds SM Colegate, PR Dorling) pp. 393–398. (CAB International: Wallingford, UK)
Elson R (2002a) ‘Code of practice for minimisation of the risk of corynetoxins contamination of hay and straw for export.’ (Department of Agriculture, Fisheries and Forestry – Australia, and Australian Quarantine and Inspection Service: Canberra)
Elson R (2002b) ‘Sampling procedures for minimization of the risk of corynetoxins contamination of hay and straw.’ (Department of Agriculture, Fisheries and Forestry – Australia and Australian Quarantine and Inspection Service: Canberra)
Gwynn R, Hadlow AJ (1971) Toxicity syndrome in sheep grazing Wimmera ryegrass in Western Australia. Australian Veterinary Journal 47, 408
| Toxicity syndrome in sheep grazing Wimmera ryegrass in Western Australia.Crossref | GoogleScholarGoogle Scholar | 5106801PubMed |
Jacobs SWL (2001) The genus Lachnagrostis (Gramineae) in Australia. Telopea 9, 439–448.
Masters AM, Gregory AR, Evans RJ, Speijers JE, Sutherland SS (2006) An enzyme-linked immunosorbent assay for the detection of Rathayibacter toxicus, the bacterium involved in annual ryegrass toxicity, in hay. Australian Journal of Agricultural Research 57, 731–742.
| An enzyme-linked immunosorbent assay for the detection of Rathayibacter toxicus, the bacterium involved in annual ryegrass toxicity, in hay.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XmvFaktbw%3D&md5=110a002d712476895723ea1593bb2860CAS |
McIntosh GH, Rac R, Thomas MR (1967) Toxicity of parasitised Wimmera ryegrass, Lolium rigidum, for sheep and cattle. Australian Veterinary Journal 43, 349–353.
| Toxicity of parasitised Wimmera ryegrass, Lolium rigidum, for sheep and cattle.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaF1c%2FhtVGqsw%3D%3D&md5=0afd83a403bc7638af1b97bd31fa6766CAS | 6069455PubMed |
McKay AC, Ophel KM (1993) Toxigenic Clavibacter/Anguina associations infecting grass seedheads. Annual Review of Phytopathology 31, 151–167.
| Toxigenic Clavibacter/Anguina associations infecting grass seedheads.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1cvms1ertg%3D%3D&md5=c8e226734b17abc6e831168d13070acaCAS | 18643766PubMed |
McKay AC, Ophel KM, Reardon TB, Gooden JM (1993) Livestock deaths associated with Clavibacter toxicus/Anguina sp., infection in seedheads of Agrostis avenacea and Polypogon monspeliensis. Plant Disease 77, 635–641.
| Livestock deaths associated with Clavibacter toxicus/Anguina sp., infection in seedheads of Agrostis avenacea and Polypogon monspeliensis.Crossref | GoogleScholarGoogle Scholar |
Riley IT, Ophel KM (1992) Clavibacter toxicus sp. nov., the bacterium responsible for annual ryegrass toxicity in Australia. International Journal of Systematic Bacteriology 42, 64–68.
| Clavibacter toxicus sp. nov., the bacterium responsible for annual ryegrass toxicity in Australia.Crossref | GoogleScholarGoogle Scholar |
Sasaki J, Chijimatsu M, Suzuki K (1998) Taxonomic significance of 2,4-diaminobutyric acid isomers in the cell wall peptidoglycan of actinomycetes and reclassification of Clavibacter toxicus as Rathayibacter toxicus comb. nov. International Journal of Systematic Bacteriology 48, 403–410.
Schneider DJ (1981) First report of annual ryegrass toxicity in the Republic of South Africa. The Onderstepoort Journal of Veterinary Research 48, 251–255.
