Recent developments in diagnosis of exotic disease at the Australian Animal Health Laboratory
AJ FORMAN CSIRO Australian Animal Health Laboratory, Geelong, Victoria 3220
Introduction The scientific program at AAHL has been active for about nine
years. Until mid-1985, the procedures for microbiological con- tainment at the Laboratory were incomplete and work on endemic diseases only was undertaken at AAHL. From that time, the development of diagnostic tests for exotic pathogens was commenced.
The emphasis in the first instance was on foot-and-mouth disease 0). Because of the decision not to import live FMD virus, work was conducted overseas to develop tests that could be adopted at AAHL, using inactivated antigens. Other work proceeded with exotic agents that were available already within Australia, having been previously imported or isolated by other laboratories. These agents included rabies virus, classical swine fever (hog cholera) viruses, Aujeszky's disease virus, Newcastle disease viruses, avian influenza virus and the agent of contagious bovine pleuropneumonia. A program of bluetongue research into improved diagnostic methods also proceeded, using virus isolated within Australia. The role of AAHL is to provide a comprehensive service for
diagnosis of the major livestock diseases exotic to Australia To f f l i l this role, further development of the diagnostic capabilities of the Laboratory was required, which was contingent on the importation of other viruses. To this end, the Armstrong Committee was appointed in 1988 to review AAHL's require- ments for exotic pathogens. The recommendations of this Committee enabled AAHL to undertake the importation of a wider range of pathogens, to increase the number of diseases for which diagnostic testing could be undertaken and to introduce procedures which enable a more rapid diagnosis to be made. Developments since that time are summarised below.
Foot-and-mouth disease and other vesicular diseases A project undertaken at the Institute for Animal Health,
Pirbright, UK in 1984/85, provided AAHL with an antigen- detectionELISAforrapiddiagnosis of the disease, using samples collected from clinical cases. Subsequently, AAHL has adopted a procedure for testing sera by ELISA with inactivated antigen, using reagents purchased from Pirbright. This is now used routinely for testing of livestock imported from FMD-infected countries and is available for investigation of disease outbreaks within Australia, should the need arise. FMD research undertaken by AAHL staff in Thailand over a
period of six years from 1986, has provided AAHL with the expertise to characterise strains of FMD virus. The technology is not yet available for this to be performed at AAHL, because a range of live viruses is required. However, we can anticipate that over the next five to ten years, alternative procedures not depend- ent on live viruses will enable AAHL to have a much better capability in this respect.
As a consequence of the recommendations of the Armstrong Committee, viruses were imported and diagnostic development undertaken for swine vesicular disease, vesicular stomatitis and vesicular exanthema of swine, all of which can mimic FMD. ELISAs are available for rapid diagnosis of clinical cases of swine vesicular disease, which occurs in pigs only, and vesicular stomatitis, which can occur in a number of species, commonly in cattle but also in horses. Vesicular exanthema of swine, which affects pigs only, is less amenable to rapid diagnosis by ELISA, and virus isolation procedures are relied upon. Antibody detec- tion tests for all of these diseases have been developed using serum neutralisation.
It is expected that in most circumstances, a provisional diag- nosis of FMD or other exotic vesicular disease, or exclusion of
Australian Veterinary J o d Vol. 70, No. 5, May 1993 161
them in a negative case, could be achieved within 24 to 48 hours of receiving a specimen.
Bluetongue Ten years ago, bluetongue serology was carried out by comple-
ment fixation or immunodiffusion tests. Both tests had deficien- cies and caused constant problems with livestock exports, with cross-reactions causing rejection of otherwise suitable animals, and variability in testing procedures sometimes causing animals to be refused at the destination country. Developments in ELISA producedanimproved test, which inrecentyearshasbeenrefined at AAHL, with recombinant antigen and monoclonal antibodies providing highly defined reagents, to be one of the most reproducible of serological assays. The final step will be to gain wide international acceptance of the test, and this is well on the way to being achieved through Office International des Epizooties (OIE). In the last three to four years a major scientific advance has enabled the practical application of in-vitro replica- tion of DNA (polymerase chain reaction, or PCR, technology) to diagnosis of disease. This has been applied to the detection of bluetongue virus at AAHL in diagnostic situations and can also be used for serotype determination and topotype (geographical origin) analysis. This work will be advanced further when bluetongue virus strains are obtained from South Africa. Testing of these strains is currently being undertaken, before they can be imported. However, the group-specific PCR has enabled the time taken for detection of bluetongue virus to be decreased from 10 days or even several weeks to one or two days.
Hog cholera and African swine fever Virus isolation, antigen detection and serology procedures have
been developed over the last five years at AAHL for hog cholera (classical swine fever). A significant improvement in rapid diag- nosis was made in the development of a rapid antigendetection ELISA, by collaboration between scientists at the Elizabeth Macarthur Agricultural Institute in New South Wales and staff at AAHL. The procedure was validated by Dr AD Shannon from EMAI, working in Holland for a time to test the assay with a range of viruses not available in Australia. This assay enables detection of virus in a specimen to be made within a few hours from receipt of suitable specimens rather than several days if cell culture isolation was the only option. Work is continuing with classical swine fever at AAHL, especially to develop improved antibody assay procedures.
The Armstrong Committee recommended the importation of a non-pathogenic strain of African swine fever (ASF) virus, and recognised that there may be a need to consider the importation of an additional, pathogenic strain to develop diagnostic tests. In the last two years procedures have been developed using the non-pathogenic strain, but further work is required using a pathogenic virus that demonstrates the characteristic of haemad- sorption, an important diagnostic feature. Approval was sub- sequently obtained to import an additional strain of the virus from Plum Island, and this has recently been received. The ability to confidently undertake tissue culture isolation for ASFvirus could reduce the time to arrive at a diagnosis from perhaps a week to probably three to four days.
Newcastle disease and avian influenza A major project of research into improved diagnosis of
Newcastlediseasehas resulted in arangeof proceduresbecoming available over the last five years. An antibody ELISA. validated in Australia and overseas, is a valid alternative to the haemag- glutination-inhibition test. Procedures developed for pathogenicity testing of field viruses, of importance in Australia
to discriminate pathogenic, exotic viruses from the non- pathogenic, endemic strains, have been applied to field problems in which there was a concern about NDV involvement. These procedures produce faster (hours or a few days) results than the defintive, but time consuming, embryo or bud pathogenicity studies that may take weeks to arrive at a final result. Tests include the use of monoclonal antibodies to determine the lo- cation of virus in inoculated embryos, the use of PCR to amplify key sequences of the genome, and the use of antibodies prepared against particular amino acid sequences, to place the virus in a pathogenicity grouping. As well as beiig a faster approach, these new procedures for pathotyping have the potential to be trans- ferred into State laboratories for application there during an outbreak.
Demands placed on laboratory staff during the 1985 outbreak of avian influenza near Bendigo, exposed the need for an antigen detection procedure to avoid the need for virus isolation attempts on large numbers of specimens. With industry fundmg, im- munofluorescence and immunoperoxidase procedures were developed at AAHL to detect viral antigen in the tissues, espe- cially pancreas, of infected birds. This assay was used with great benefit in the 1992 outbreak, both at the Bendigo laboratory and at AAHL and has greatly increased the speed with which suspect flocks can be screened for possible infection, from days to hours. An ELISA for antibody detection, which was also developed, is in routine use for the testing of birds being imported into Australia. Procedures have been developed and applied for the determination of potential pathogenicity of avian influenza viruses using PCR. Genomic analysis is part of the internationally agreed procedure for defining the pathogenicity of isolates. Although the method can now be applied for H7 viruses (involved in the three Victorian outbreaks), it has not yet been developed forotherstrains,for whichAAHLdoesnot havepathogenic avian representatives. Such procedures can reduce the time taken to establish pathogenic potential of an isolated virus from the 10 days required for a bud inoculation procedure, to five or six days, and work is continuing to further reduce the time required.