Establishment of improved methods for the diagnosis and control of livestock diseases in south-east Asia using enzyme linked immunoabsorbent Assay (Elisa)

• Universiti Pertanian Malaysia, Malaysia
• Research Institute for Veterinary Science, Indonesia

Many Southeast Asian countries have facilities for livestock disease diagnosis - usually a centralised laboratory or research station and outlying smaller ones. These service a wide range of mammalian, avian and fish enterprises at smallholder and commercial levels, reflecting the importance of these livestock industries. Animals in these countries provide milk, meat and draught power, and represent individual or community wealth.

Enzyme-linked Immunosorbent Assay (ELISA) systems for diagnosing and controlling animal diseases are inexpensive, sensitive and versatile, and can readily be adapted to a wide variety of local requirements. This project seeks to utilise, modify and develop ELISA-based systems, within the existing government framework, in a number of Southeast Asian countries - initially Indonesia and Malaysia.

Within Indonesia, the scientists will develop ELISA for the following purposes: laboratory diagnosis of Brucella abortus in cattle and buffalo, of B. suis in pigs and of leptospirosis in cattle, buffalo and pigs; field and laboratory diagnosis of haemorrhagic septicaemia in all three animals; the field diagnosis of anthrax. In Malaysia, they will develop ELISA for the laboratory diagnosis of Newcastle disease in village poultry. Major goals will be to extend the availability of ELISA technology and expertise to regional laboratories in both countries, and to form a basis for a network of ELISA capability (and co-ordinate and standardise the supply of specific reagents) throughout the region.

Tests to diagnose the different forms of brucellosis have employed numerous antigens. The team will refine existing ELISA methods and extend these to the detection of B. suis antibody in pigs, emphasising the reproducibility and standardisation of the testing procedures. Refinements include the use of protein A-enzyme conjugate as the detecting agent in lieu of anti-globulin-enzyme conjugates. The team will also try to produce an ELISA capable of detecting Brucella in clinical samples as an alternative to costly and time-consuming bacteriological culturing.

In ELISA systems for serological diagnosis of leptospirosis, the main drawback to date has been the lack of serovar specificity. Standard immunoblotting and affinity chromatography techniques will be used to characterise serovar-associated antigens in order to develop specific ELISA systems - initially for hardjo and pomona, with eventual extension to other important serovars.

The team will characterise antigen preparations from Pasteurella multocida (the causal agent in haemorrhagic septicaemia) for specificity and test them by ELISA methods. After raising specific antisera in experimental animals, they will conjugate these to horseradish peroxidase, assess an ELISA for P. multocida in the laboratory and extend this assessment to the ability to detect organisms in the saliva of clinically affected animals.

For the anthrax application, having first produced bulk cultures of Bacillus anthracis the scientists will characterise antigens, particularly the capsular antigens, using polyacrylamide gel and immunoblotting techniques, and produce antisera to strain-specific antigens in experimental animals. They will then isolate antibody fractions, conjugate these to horseradish peroxidase and assess an ELISA for anthrax in the laboratory. Field evaluations will compare the test with conventional methods.

In Malaysia, virus isolated from field outbreaks of Newcastle disease, and attenuated vaccine strains, will be purified by ultra-centrifuging and characterised using polyacrylamide gel electrophoresis and immunoblotting. The scientists will utilise suitable antigens in an ELISA to detect specific antibody in known vaccinated and infected birds, and conduct correlation studies with the current haemagglutination inhibition assay and infectivity status. Standardised ELISA test kits will be prepared.

An important component of the project will be a 'hands-on' workshop late in the second year. It will primarily serve scientists from Southeast Asia, while others from the western Pacific and Australasia may also attend. It will act as a focus for the formation of an ELISA 'network' centred on these three regions.

Development of these ELISA testing systems will allow the design of practical disease control and/or eradication programs in Southeast Asia as well as epidemiological surveys and economic evaluation. In addition, all these diseases also occur in Australia and the work in Southeast Asia - where their higher prevalence will make the task of standardising the tests easier - will have benefit here.