The principal research streams in this thesis were tickborne viruses of terrestrial and sea birds, Culicoides transmitted viruses of livestock and mosquito borne rhabdoviral infections of cattle in Australia.
Five viruses new to science in Australia or the world were isolated from ticks associated with seabirds in the period covered by this thesis. Precarious Point virus (an Uukuvirus); Gadgets Gully virus (a flavivirus); and a third virus, CSIRO 92, were isolated from Ixodes uriae ticks collected from Penguin colonies on MacQuarie Island in the subantarctic. Another virus (CSIRO 1568) (provisional name Albatross Island virus) from Ixodes eudyptidis collected from Albatross Island in Bass Strait; and CSIRO 1759 virus from Ixodes kohlsi collected from a Little Penguin colony on Little Diamond Island, Bicheno, Tasmania. A virus, designated CSIRO 1499 virus, also new to Australian science, was isolated from Ixodes robertsi collected from a Cattle Egret rookery in Queensland. The isolations were made in BKH21 tissue cultures instead of suckling mice, as was the standard method in Australia and other countries at the time.
A sentinel cattle herd network with an associated system of insect vector trapping and identification was refined to study the ecology of bluetongue and other arboviruses infecting livestock throughout Australia. This system has been formally adopted by the Australian Quarantine Inspection Service as a means of monitoring the ecology of endemic arboviruses infecting livestock and an early warning system for intrusions of presently exotic arboviruses and insect vectors. Sentinel herd surveillance is used as a means of gaining and maintaining zonal freedom from specific arboviral diseases to enable live animal export. The serological data for 16 arboviruses was transferred into electronic form for potential future use in certification.
Bluetongue, a disease of sheep, is carried as a silent infection of cattle. The distribution of bluetongue serotypes 1 and 21 in Australia was found to be limited to the north and east of the continent. These serotypes are much more widely distributed than the other eight serotypes that have occurred from time to time. Bluetongue rarely occurs as a natural disease in Australia. The bluetongue viruses that circulate silently in cattle are usually pathogenic for Merino sheep, but the pathogenicity declines on further sheep to sheep transfer of viruses. The knowledge of the ecology of bluetongue viruses and their vectors has been used to coordinate research into this infection in Australia. The techniques developed here were later used to design and partially implement a project which allowed for the isolation of viruses of 12 bluetongue serotypes in China, thus facilitating export of live cattle and sheep to that country from Australia.
Four of the five serotypes of epizootic hemorrhagic disease (EHD) were isolated for the first time in Australia, EHD2, 5, 6 and 7. Together with a fifth Australian serotype, EHD8, they were compared with the other known EHD viruses in the world and found to be distinct. Serological surveys were also carried out for EHD 2 and 5. Their distribution in cattle is limited to northern and eastern Australia and is consistent with Culicoides brevitarsis being a vector. EHD5 was isolated from this midge. In contrast, the orbivirus, Stretch Lagoon Virus was isolated from a mosquito Culex orbostiensis.
Aino, Peaton, Tinaroo and Douglas viruses, all of the Simbu group, were also found to have a substantially northern and easterly distribution, with southern Australia substantially free of all of this group of viruses. The cross-sectional surveys and sentinel herd seroconversions are consistent with Culicoides brevitarsis being the major vector. Aino, CSIRO Village and Bunyip Creek viruses were isolated from this midge for the first time, thus strengthening the association. The results of the distribution of these arboviruses, plus others, have defined the theory that they are intrusions from Indonesia and not Papua-New Guinea. There is a high probability that additional arbovirus infecting cattle will continue to blow in within infected midges.
The number of rhabdoviruses, known to infect cattle in Australia has grown from one (bovine ephemeral fever virus) at the commencement of investigations into the epidemiology and pathogenesis of ephemeral fever by my research group, to six. Of these, bovine ephemeral fever virus, Kimberley, Adelaide River and Berrimah viruses have formed an antigenic group, now known as ephemeroviruses. Tibrogargan and Coastal Plains virus also infect cattle, but their relevance to ephemeral fever or any other disease is unknown. Oak Vale virus, another rhabdovirus, has been isolated from Culex edwardisi and C. austropalpalis. Its significance as a disease agent is unknown. An additional rhabdovirus Walkabout Creek was isolated from C.Austropalpalis.
The epidemiology of ephemeral fever in Australia was shown to have altered from a spaced north-south movement through the cattle of eastern Australia, prior to 1975, to continuous smaller local epidemics, resembling endemicity. Evidence for the interference with the epidemic spread of BEF virus, by at least one unrelated arbovirus (CSIRO Village virus), was discovered. Also, the effect of antibodies, heterotypically generated by Kimberley and Adelaide River viruses, on the length of viraemias in ephemeral fever was discovered. Bivine ephemeral fever is confirmed as a biphsic fever with a time lapse of 102.6 ± 9.5 hours from virus injection into the bloodstream to the onset of severe clinical signs.
Pathogenesis studies, and the demonstrated absence of BEF virus from the lymphatic system of cattle during disease, support the role of mosquitoes as vectors. This accords with the necessity to inject pathogenic BEF virus into veins to produce experimental disease. In spite of BEF virus being isolated from C. brevitarsis, it is highly probable that this midge is not significant in the transmission of BEF virus, as its mouth parts are not inserted into a venule during feeding.
The fundamental nature of a generalized inflammation of the vascular system, joints and serous spaces, has been confirmed by serial biochemical studies of natural and experimental ephemeral fever. The haematology is very characteristic with a neutrophilia overshadowing a lymphopaenia and an eosinopaenia. There was no change in the values of creatinine, urea, gamma glutamyl transferase, aspartate amino transferase or alkaline phosphatase. The main biochemical changes are those accompanying inflammation. They include a hyperglycaemia, followed by a hypoglycaemia, with a reciprocal hypophosphataemia. Plasma zinc and iron levels fall. Plasma fibrinogen rises rapidly. Reduced plasm calcium levels occur on the first day of disease in step with muscular tremor or fasiculation, loss of gastrointestinal mobility and the swallowing reflex and other clinical signs of hypocalcaemia paresis or paralysis.
Treatment of hypocalcaemic signs in ephemeral fever with calcium borogluconate produced a response within minutes. Treatment with phenybutazone, an anti-inflammatory drug, was successful when given after clinical signs were observed. If given in advance of the expected appearance of disease, fever and all visible clinical signs were prevented. Continuous treatment for the expected course of the disease prevented clinical signs, but not the development of immunity to subsequent challenge with virulent BEF virus. This means that the clinical signs are not caused directly by the virus. Viraemia was reduced in length, probably because of the reduction of inflammation and consequent neutrophil response. A second anti-inflammatory drug, flunixin meglumine, elicited a rapid resolution of fever for 5-14 hours, but not of all clinical signs.
The presence of interferons in the plasma was detected after viraemia commenced, but before fever and other clinical signs began in experimental cases. The level of interferon declined before fever resolved in experimental and in natural disease. The pattern of appearance and decline was unaffected by treatment with phenylbutazone. An hypothesis was advanced that interferon toxicity is responsible for the intravascular inflammation that produces many of the clinical signs of ephemeral fever.
A program of serial blood sampling, using the same techniques as for ephemeral fever, was applied to multiparous cows in the periparturient period. One of four such cows developed milk fever. The biochemistry of the plasma changed in a similar fashion to that of ephemeral fever. Hypocalcaemia occurred as in ephemeral fever. A neutrophilia and an eosinopaenia occurred close to calving. The lymphocyte numbers rose in the periparturient period of the cows with and without milk fever in contrast to the lymphopaenia of ephemeral fever. A single heifer was shown to have normal haematology spanning parturition in contrast to multiparous cows. An hypothesis is advanced that inflammation, antigen/antibody complex, plus plasma pH rise play parts in the initiation of hypocalcaemia of multiparous cows by suppressing the ionized calcium level in the blood.
The temporary hind-quarter paralysis, characteristic of many cattle with severe ephemeral fever, has been separated into two components. One is caused by hypocalcaemia and is responsive to calcium replacement therapy. The other component probably is related to a loss of myelin in the upper cervical part of the spinal cord.
More efficient methods of isolation of BEF virus in Aedes albopictus tissue cultures were developed and demonstrated a viraemia which varied from 28 to 156 hours. An ELISA test, using a monoclonal antibody, was developed and shown to be more accurate and specific than the established virus neutralisation test. An antigen detection system was found to detect a similar length of viraemia as the virus isolation system.
A collection of strains of BEF virus from natural cases between 1956 and 1992 was analysed with a library of monoclonal antibodies. Considerable antigenic variation was found. A limited test in cattle with Australian and Chinese strains of virulent BEF virus, showed complete cross protection against challenge, after one episode of disease on the two strains, in spite of antigenic variation. A sub-unit vaccine was developed from the G protein of an Australian strain of BEF virus (BB7721). A similar vaccine was produced in China to the Beijing 1 strain. The vaccines were cross protective.
In 1976, in conjunction with the Director of Queensland Institute of Medical Research, the Australian Arbovirus Research in Australia Symposia and associated proceedings were inaugurated. Eleven symposia have been held with the most recent held in August 2012. Similar symposia on bovine ephemeral fever and bluetongue were conducted in China with derivative proceedings. Both these symposia brought together the Chinese scientists working on these diseases for the first time and demonstrating their knowledge to the world.