SPECIAL ARTICLE

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

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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.

Rinderpest and peste des petits ruminants Rinderpest virus was imported as live, attenuated vaccine.

Stocks of the vaccine are held by CSL Ltd for release to quaran- tine authorities for the vaccination of livestock for export to the Middle East, under strictly supervised circumstances. The vac- cine stock was used at AAHL to propagate virus that was then used to develop an antibody assay procedure by serumneutralisa- tion. Rinderpest and peste des petits ruminants are very closely related, so that this assay could be applied to either disease. The attenuated virus, being a tissueculture adapted virus, grows readily in culture. There is no opportunity for AAHL to verify virus isolation procedures from clinical specimens as the attenuated virus does not cause disease in livestock. The Armstrong Committee recommended against the importation of a virulent strain of rinderpest virus.

Scrapie and bovine spongiform encephalopath y It was recognised in 1991 that a capability to detect the agent

causing scrapie and bovine spongiform encephalopathy (BSE) would be required for confiiatory negative testing of sheep imported into Australia under the Scrapie Freedom Assurance Program, before release of their progeny from quarantine. In addition, this country would benefit from AAHL being able to test suspicious cases of scrapie or BSE, rather than having to send themoverseas. Amouse-adapted scrapie agent was imported into AAHL toward the end of 199 1, and procedures have been applied for the detection of scrapie-associated fibrils by electron microscopy. The procedure has already been used in a diagnostic

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situation, to exclude scrapie in a suspect animal within two days, compared with a delay of about one week to have a specimen examined overseas.

Rabies The Armstrong Committee recommended that rabies-related

viruses (Duvenhage, Obodhiang, Kotonkan, Lagos bat and Mokola) be imported into AAHL to extend the capability for distinguishing strains of virus that may be isolated from cases of the disease. The viruses have been imported but have not been used to date. The Centers for Disease Control, Atlanta, provided fluorescein-tagged monoclonal antibodies that enable a virus to be identified as the rabies virus family and as either classical rabies or one of the related viruses. It is believed that this is as far as AAHL should need to go in characterising an isolated rabies virus. CDC maintains a service worldwide for the detailed characterisation of rabies virus isolates, and it is appropriate for AAHL to avail itself of this service, as was done with a human isolate in 1991 (McColl er ul 1993). A change from mouse inoculation to the use of tissue culture (mouse neuroblastoma cells) for isolating virus has led to a reduction in time for com- pletion of exclusion testing, from 30 days to two days.

African horsesickness and equine influenza After importation of representative viruses of the nine serotypes

of Africanhorsesicknessvirus in 199 1,virus isolation procedures were developed and an ELIS A for antibody detection established. All of the viruses are attenuated and cannot produce disease in horses. Consequently, validation of virus isolation and antigen detection procedures will not proceed any further.

In 1987, inactivated antigens of the two classical equine influen- za viruses were obtained to enable AAJ3L to have a basic capability to undertake serology. The Australian horse industry recognises influenza as a signifcant threat, and in 1991 initiated proposals for AAHL to import viruses to improve its prepared- ness by developing a capability to isolate virus, detect antigen and carry out antibody assays. Approval was obtained in October 1992 to import these viruses, and additional development work is in progress.

Porcine reproductive and respiratory syndrome Porcine reproductive and respiratory syndrome (PRRS) is a

newly recognised disease of pigs, which appears to have spread in the last two years from North America into Europe. A proposal to import strains of the virus and develop diagnostic procedures was approved in October 1992, and development of tests is proceeding. Concerns about the introduction of this virus into Australian pig herds, through importation of Canadian pig meat, have led to more stringent controls being placed on that trade. Australia will probably come under some international pressure to substantiate its claim to freedom from PRRS in order to justify its import restrictions on pig products. Accordingly, staff at AAHL have given a high priority to the development of a serological test for PRRS viral antibody, to enable a survey of Australian pigs to be undertaken.

Other diseases Rift Valley fever virus is a human pathogen and requires special

considerations for staff working with live virus. Serology can be undertaken at AAHL using imported, inactivated reagents. The live virus was obtained in 1991 and work will proceed to validate virus isolation procedures.

Epizootic haemomhagic disease of deer, which is of little con- cern in Australia as a disease of livestock, is a constraint on livestock exports to some countries, so that antibody testing is a requirement. A competition ELISA, of a similar format to the ELISA for bluetongue, has been developed and is being validated.

The virus of malignant catarrhal fever, which causes sporadic disease in cattle in Australia, cannot be grown in cell culture. This prevents serology W i g undertaken, which has inhibited the ability of investigators to understand the epidemiology of the disease. The same disease in Africa is caused by a virus that does grow in cell culture. This virus has been imported into AAHL and used to develop a serum neutralisation test that has been used to investigate field problems. Three strains of maedi-visna virus were imported into AAHL

in 1991. One strain has been used to develop an ELISA for antibody detection and to optimise cell culture procedures for isolating the virus. Work is continuing with the other strains to ensure that procedures are applicable to all strains.

Conclusions The recommendations of the Armstrong Committee in 1988 and

the AAHL Programs and Priorities Review in 1990, have enabled AAHL to greatly expand the number of diseases for which a diagnostic service can be provided, For most diseases, there are always improvements that can be made in sensitivity and specificity of tests. In particular, many of the procedures used at AAHL have not been rigorously validated, because of the lack of clinical material with which to test them and a lack of an appropriate range of strains of a virus, to confirm that an assay will provide reliable results with a variety of isolates. Over the next one or two years, AAHL, will have largely developed tests as far as is practicable with the Viruses available, and the major limitations of them will result from the inability to validate tests with clinical specimens generated in experimental animals using pathogenic viruses of a variety of strains. There will be a continu- ing need to import a greater range of Viruses into AAHL, or to seek further opportunities for AAHL staff to undertake research overseas to improve the quality of their diagnostic systems.

References Australian Animal Health Laboratory (1990) Programv and Priorities

Review:aReporttothe Board of the Auriralian Animal Healthkboratory. AAHL, Geelong, Victoria

Committee to Review the Australian Animal Health Laboratory’s Requirements for Exotic Pathogens (Armstrong Committee) (1988) Report. AGPS, Canbema

McColl KA, Gould AR, Selleck PW, H q x r PT. Westbury HA and Smith IS (1993)Awt VetJ7084

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