In Indonesia, Bali cattle (Bos javanicus) have been distributed extensively to smallholder farmers throughout the country under several development programs. The cattle are owned almost exclusively by smallholders and improve integrated farming systems, primarily through their use as draught animals, as well as in meat production, the consumption of crop residues for fodder, and the contribution of their manure to soil fertility.
Bali cattle have a high susceptibility to Jembrana disease - a viral disease detected only in Indonesia, and endemic in Bali, Kalimantan, Java and Sumatra. (The causative virus is referred to as Jembrana Disease Virus or JDV.) This disease was first detected in 1964 in Bali cattle (on the island of Bali itself) where it killed many thousands of animals. As a result a ban on inter-island trade of cattle was introduced to affected areas. This ban consequently affected food production, especially on the eastern islands. Jembrana disease, therefore, is a major threat to the success of the various Bali cattle distribution programs and consequently the attempts to alleviate poverty and increase food production in Indonesia.
Recent research partly supported by ACIAR has demonstrated that a Jembrana disease vaccine is possible. The effective vaccine was derived from tissues of infected animals. However, despite its efficacy, it has serious disadvantages that make its widespread use in Indonesia inadvisable and impractical: it is too expensive to produce, it is unstable, and due to the nature of the vaccine, adequate quality control is at best difficult and probably ultimately impossible. It is not possible for Indonesia to utilise a vaccine that has been developed elsewhere, due to the disease being largely in Bali Cattle.
This project aimed to improve the diagnosis of Jembrana disease, and to develop a vaccine that is safe, effective and of lower cost than the current one.
Three possible approaches for an improved vaccine were investigated:
The use of antigenically cross-reactive bovine immunodeficiency virus (BIV) as a heterologous vaccine;
The use of viral proteins produced by recombinant DNA technology;
"Naked" DNA vaccines, which have the advantage of a relatively simple production process, stability, and no requirement for refrigeration. (However, no DNA vaccines were currently in commercial use.)
A parallel strand of the work studied ways to improve the diagnosis of Jembrana disease in provincial Disease Investigation Centres (DICs) throughout Indonesia. This was supported by the development of ELISA assays for the detection of antibodies, and in situ hybridisation techniques for the detection of virus in tissues of animals suspected of dying from the disease. The techniques were deemed suitable for use in provincial laboratories throughout Indonesia.
While diagnostic and immunosurveillance techniques were developed and transferred to provincial diagnostic laboratories throughout Indonesia in the first two years of the project, some improved capability was achieved in the third year. First, to facilitate the diagnosis and future research associated with Jembrana disease, a range of monoclonal antibodies were produced against the proteins of Jembrana disease virus. The hybridomas secreting these monoclonal antibodies are now securely stored at Murdoch University. Second, an immunoperoxidase test was developed to detect JDV in peripheral blood lymphocytes. This test enabled the diagnosis of Jembrana disease as early as 4 days after infection and has advantages over previous methods.
There is a critical need to define the non-pathogenic bovine lentivirus present in Sulawesi. This virus, which is very closely related to Jembrana disease virus, will interfere with the diagnostic methods, in the event of an outbreak of Jembrana disease in Sulawesi. It will be necessary to transmit the infection to donor cattle in Denpasar in order to conduct investigations. In 2003-2004, these attempts failed. Attempts to detect bovine lentivirus DNA in the antibody-positive cattle in Sulawesi have also been unsuccessful. Attempts to develop a serological test that will differentiate antibody to the pathogenic Jembrana disease virus and the non-pathogenic Jembrana disease-like virus have also continued but without success.
An extensive examination of the genome of Jembrana disease virus isolates from throughout Indonesia was undertaken during 2003-2004. The results indicated a small variation only in gag and env regions of virus strains from Bali, Lampung and Kalimantan, limited sequence divergence over a 15 year period, and some geographical divergence (greater differences in the strain from Kalimantan than the intra-strain variation in Bali samples). It appears that Jembrana disease virus, unlike other lentiviruses, exhibits remarkable conservation in its genome; it has varied only little in the past 15 years. A positive benefit of this is that diagnostic techniques developed using molecular biological approaches should be effective against all strains throughout Indonesia. Recombinant proteins developed against any strain should provide effective antigens for immunosurveillance techniques for all viruses, and should also provide effective vaccines for use throughout Indonesia.
Extensive developmental work was conducted at Murdoch University to produce recombinant proteins that could be used as vaccines. The proteins produced included a truncated SU, Capsid, and Tat protein. Extensive testing of these recombinant proteins as potential vaccines was conducted during a series of 4 trials involving 24 cattle each experiment. The results of the vaccine trials are very promising; they indicate that vaccination with SU and Tat reduced the duration of the febrile period and virus load. Further clinical trials are therefore warranted using the recombinant SU and Tat vaccines, probably a combination of both.
Four potential DNA vaccine constructs were prepared and tested for their ability to produce protein in vitro. Two of the constructs pVAX/tat and pVAX/capsid have been demonstrated to produce protein in vitro and were considered worthy of in vivo evaluation as potential DNA vaccines for Jembrana disease. A small pilot animal trial was conducted at the Disease Investigation Centre Denpasar to test the effectiveness of these vaccines. Immunisation with these constructs did not induce a protective immunity but optimal methods of administration of the vaccines were not achieved.
In view of the success achieved by vaccination with recombinant proteins, lack of success with the small scale DNA experiment in cattle, and limited time and funding, future work should be directed toward the use of these proteins as vaccines. A major need will be to find an Indonesian vaccine company willing and able to utilise the recombinant DNA technology for the production of commercial quantities of vaccine.
Links:
[1] http://www.aciar.gov.au/country/Indonesia
[2] http://www.aciar.gov.au/programarea/Animal Health