Post-viraemic detection of bovine ephemeral fever virus by use of autogenous lymphoid tissue-derived bovine primary cell cultures.

BACKGROUND: The potential tissue replication sites and specific cell types that support in vivo virus survival beyond the acute phase of bovine ephemeral fever virus (BEFV) infection have not been fully defined in cattle. To clarify the knowledge gap, tissue specimens were tested after collection from an adult steer necropsied 1 week after acute BEF. CASE REPORT: Significant necropsy findings included fibrinoproliferative synovitis in the stifle joints and fibrin clot-laden fluid in serous body cavities. Moderate numbers of infiltrating neutrophils were demonstrated in sections of the prefemoral lymph nodes and haemal node, and lymphoid hyperplasia in the spleen, haemal node and prefemoral lymph nodes. Viral RNA was detected by qRT-PCR in fresh spleen, haemal node, prefemoral lymph node, synovial fluid and in several spleen-derived cell cultures. BEFV was isolated from autogenously derived splenic primary cell cultures 6 days after cessation of viraemia, and characteristic bullet-shaped virions were confirmed by electron microscopy of an ultrathin haemal node section. In sections of the spleen, haemal node and other tissues, immunohistochemistry demonstrated BEFV antigens that were intracellularly associated with probable histiocytic cells. CONCLUSION: BEFV has preferential tropism for bovine lymphoid tissues and the spleen and haemal node may be potential sites for post-viraemic virus replication.

Viral neurotropism, peripheral neuropathy and other morphological abnormalities in bovine ephemeral fever virus-infected downer cattle.

OBJECTIVE: This study assessed the neurotropism of bovine ephemeral fever (BEF) virus (BEFV) and described histomorphological abnormalities of the brain, spinal cord and peripheral nerves that may causally contribute to paresis or paralysis in BEF. METHODS: Four paralysed and six asymptomatic but virus-infected cattle were monitored, and blood and serum samples screened by qRT-PCR, virus isolation and neutralisation tests. Fresh brain, spinal cord, peripheral nerve and other tissues were qRT-PCR-tested for viral RNA, while formalin-fixed specimens were processed routinely and immunohistochemically evaluated for histomorphological abnormalities and viral antigen distribution, respectively. RESULTS: The neurotropism of BEFV was immunohistochemically confirmed in the brain and peripheral nerves and peripheral neuropathy was demonstrated in three paralysed but not the six aneurological but virus-infected animals. Wallerian degeneration (WD) was present in the ventral funicular white matter of the lumbar spinal cord of a paralysed steer and in cervical and thoracic spinal cord segments of three paralysed animals. Although no spinal cord lesions were seen in the steer euthanased within 7 days of illness, peripheral neuropathy was present and more severe in nerves of the brachial plexuses than in the gluteal or fibular nerves. The only steer with WD in the lumbar spinal cord also showed intrahistiocytic cell viral antigen that was spatially distributed within areas of moderate brain stem encephalitis. CONCLUSION: The data confirmed neurotropism of BEFV in cattle and documented histomorphological abnormalities in peripheral nerves and brain which, together with spinal cord lesions, may contribute to chronic paralysis in BEFV-infected downer cattle.

Kinetics of selected plasma cytokines during innate-adaptive immune response transition in adult cattle infected with the bovine ephemeral fever virus.

While virus neutralizing antibodies are known to be variably protective against bovine ephemeral fever (BEF) virus (BEFV) infections, the cytokine events that mediate the nascent adaptive immune response have not been defined in cattle. This study determined the plasma kinetics of IL-2, IFN-γ, IL-6, and IL-10 during the period of innate-immune response transition and evaluated the relationship between the virus neutralizing antibody response and viraemia in BEFV-infected cattle. Plasma from four virus-infected and uninfected negative control animals was tested by cytokine-specific immunoenzymatic assays, viraemia monitored by qRT-PCR, and virus neutralizing antibody titres determined using a standard protocol. Unlike the negative controls, plasma IL-6 and IL-10 were increased in all the virus-infected animals starting several days prior to initiation of viraemia. In one animal, plasma IL-2 and IFN-γ were consistently higher than in the other three virus-infected animals and the negative control mean. The animal with the strongest IL-2 and IFN-γ responses had the shortest viraemia while the heifer with the lowest IL-2/IFN-γ indices demonstrated the longest viraemia. Evidently, increase in plasma IL-6 and IL-10 precedes seroconversion during BEFV infections in cattle suggesting the two cytokines may influence immunological events that pave way to B-cell activation and seroconversion. While there is remarkable variability in IL-2 and IFN-γ expression amongst BEFV-infected animals, increased plasma levels of the two cytokines appear to be associated with a shorter viraemia. Ongoing studies will help define the precise role of T cells in anti-BEFV adaptive immune responses.

Kinetics of pro-inflammatory cytokines, interleukin-10, and virus neutralising antibodies during acute ephemeral fever virus infections in Brahman cattle.

While fever and inflammation are hallmark features of bovine ephemeral fever (BEF), the cytokine networks that underlie the acute phase of the disease have not been empirically defined in cattle. This study characterised the plasma kinetics of proinflammatory cytokines (IL-1ß, IL-6, TNF-α) and IL-10 during acute BEF and elucidated on the relationship between the onset of the virus neutralizing antibody response and resolution of viraemia in natural BEF virus (BEFV) infections in cattle. Plasma from three BEFV-infected and three uninfected cattle was tested for the study cytokines by a cELISA, viraemia monitored by qRT-PCR, and virus neutralizing antibody titres determined using a standard protocol. Unlike the negative controls, plasma concentrations of IL-1ß, TNF-α, IL-6, and IL-10 were consistently increased in the three virus-infected animals. Two of the infected heifers were recumbent and pyrexic on the first day of monitoring and increased cytokine production was already in progress by the time viraemia was detected in all the three infected animals. In all the virus-infected heifers, IL-1ß was the most strongly expressed cytokine, IL-6 and IL-10 manifested intermediate plasma concentrations while TNF-α was the least expressed and demonstrated bi-phasic peaks three and five days after the onset of pyrexia. In two of the BEFV-infected heifers, viraemia resolved on the day of seroconversion while in the other infected animal, viral RNA was detectable up to three days after seroconversion. The present data document variable increase in plasma IL-1ß, IL-6, TNF-α, and IL-10 during natural BEFV infections and the fact that upregulation of all but TNF-α precedes seroconversion. In addition to virus neutralising antibodies, it is likely that cytokine-mediated cellular mechanisms may be required for resolution of viraemia in BEF. Considering the anti-inflammatory properties of IL-10, its upregulation may potentially antagonise the fever response in BEFV-infected cattle.

Bluetongue surveillance methods in an endemic area: Australia.

Surveillance for bluetongue (BT) viruses (BTV) has been carried out in the Northern Territory, Australia since 1980. The number of sites, intensity of sampling and methods of testing have varied during this period. Monthly serology is conducted at a number of sentinel sites and intensive weekly sampling for virus isolation is conducted at the site of highest known arboviral activity. This has enabled the isolation of all eight BTV serotypes identified in Australia. Natural viraemias are between one and eight weeks. No additional serotypes have been isolated since 1986. However, genetic analysis of isolates has shown incursions of viruses of South-East Asian origin in 1992, 1994 and 1995. Trapping for Culicoides spp. has also been carried out at these sites on a regular basis. In recent years, an annual serological survey has supplemented the sentinel herds to more accurately define the BT zones described under OIE guidelines.

Protection of cattle from Culicoides spp. in Australia by shelter and chemical treatments.

Trials were conducted in three regions of Australia to investigate the potential for improvised shelters and chemical treatments to reduce feeding by Culicoides on cattle and thereby minimise the risk of bluetongue transmission during transport of cattle to ports. Various designs and combinations of roofs and walls were placed around penned cattle. Chemical treatments were applied to other penned cattle. Culicoides were collected from the cattle by vacuum samplers or by light traps in the pens. Roofs alone did not consistently reduce the numbers of Culicoides brevitarsis or C. fulvus and increased the numbers of C. actoni collected. Walls alone reduced the numbers of C. wadai but not C. brevitarsis. Roofs and walls in combination reduced the numbers of C. brevitarsis and C. wadai. The chemical treatments 'Flyaway' (a blend of repellents) and fenvalerate reduced the numbers of C. brevitarsis and C. wadai up to 52 h post treatment.

Temporal activity of biting midges (Diptera: Ceratopogonidae) on cattle near Darwin, Northern Territory, Australia.

The activity of nine species of biting midges aspirated from cattle was recorded in the late afternoon, evening and early morning at a site near Darwin, Northern Territory, between March and June in 1999 and 2001. There were no significant differences between the temporal activity patterns for nulliparous and parous females of any species. Nulliparous females dominated collections of all species except Culicoides marksi. C. actoni and Forcipomyia (Lasiohelea) sp., were mostly active during daylight hours while C. peregrinus, C. bundyensis and C. brevipalpis, were nocturnal. Differences in the peak activity of C. brevitarsis were noted between years and occurred slightly earlier than that observed at other sites. C. fulvus, C. marksi and C. oxystoma were generally crepuscular but differed in the length and peak period of activity. C. actoni was four times more active in the evening than in the morning while C. marksi and C. peregrinus, were respectively 2.6 and 3.4 times more active in the morning than in the evening. Numbers of the other six species were not significantly different in the evening and morning. All nine species were collected at least once from cattle shortly after dawn.

Genetic diversity of bluetongue viruses in Australasia.

The authors have characterised the genetic diversity of the bluetongue virus (BTV) RNA segments 3 and 10 from Indonesia, Malaysia and Australia. Analysis of RNA segment 3, which codes for the core protein VP3, showed conserved sequences in the previously defined Australasian topotype, but which further divided into four distinct clades or genotypes. Certain genotypes appeared to be geographically restricted while others were distributed widely throughout South-East Asia. Ongoing surveillance programmes in Australia have identified the movement of Indonesian genotypes into northern Australia and possible reassortment among them. Similarly, analysis of RNA segment 10, which codes for the non-structural protein NS3/3A, showed they were also conserved and grouped into five clades or genotypes, three Asian and two North American/South African.

Bluetongue virus does not persist in naturally infected cattle.

Studies were designed to test if observations by Takamatsu et al. in 2003 were applicable to natural infection of cattle with bluetongue virus (BTV). These observations suggested that ovine gamma delta T-cells could become persistently infected and subsequent midge feeding could induce virus replication. Skin biopsies and blood were collected from 28 cattle naturally infected with BTV-1. Blood samples were processed for virus isolation by embryonated chicken egg inoculation and for serology by BTV competitive enzyme-linked immunosorbent assay and BTV-1 virus neutralisation. BTV-1 was isolated from the blood of all animals and serology confirmed infection with BTV-1. A total of 288 skin biopsies were collected and cultured in the presence of interleukin 2 and epidermal growth factor. Sampling commenced as soon as either serology or virus isolation indicated infection with BTV and continued at weekly intervals for at least eight weeks then monthly for another two months. The natural viraemias in this experiment ranged from one to five weeks. BTV-1 was isolated from only one skin biopsy sample. This sample was collected during the week in which the animal was viraemic. These findings provide compelling evidence that BTV does not persist in gamma delta T-cells in the skin of naturally infected cattle.

Excretion of bluetongue virus in cattle semen: a feature of laboratory-adapted virus.

A series of experiments was conducted over a period of four years and involved both young (2-4 years) and old bulls (5-15 years) that were both naturally and experimentally infected with bluetongue virus (BTV). Several different virus serotypes were studied. In the Northern Territory, young bulls were exposed to natural infection with BTV over three wet seasons. During this time, bulls were infected with BTV-1, BTV-3, BTV-16 and BTV-20. In New South Wales, semen samples were examined from a large group of bulls of mixed ages that were naturally infected with BTV-1. Experimental infections in both young and old bulls (5-8 animals per group) employed both 'wild-type' and laboratory-adapted viruses from serotypes 1 and 23. A total of 41 bulls were included in the studies of natural BTV infection and 52 bulls in experimental infections. There was no evidence of BTV in any of the semen samples collected from naturally infected bulls or experimentally infected young bulls. BTV was detected intermittently in semen from a number of old bulls infected with both laboratory-adapted BTV-1 and BTV-23. These detections occurred during or immediately after the period of detectable viraemia. Virus was also detected in a few semen samples from very old bulls infected with 'wild-type' BTV-23. These samples were collected during the period of viraemia and there was usually evidence of blood in the semen. Viraemia varied in duration between 17 and 38 days. Following immunosuppression, there was no evidence of resurgence of viraemia, or excretion of virus in semen, even in animals in which virus had been previously detected in semen. When the bulls were slaughtered, virus was not detected in any tissues.

Characterisation and monitoring of neutralisation-resistant VP2 phenotypes in BTV-1 isolates from northern Australia collected over a twenty-year period.

Phenotypic profiles of the VP2 protein of isolates of bluetongue virus serotype 1 (BTV-1) collected from Queensland and the Northern Territory, Australia, between 1979 and 1986 were analysed using neutralising monoclonal antibodies (MAbs) raised to the prototype isolate of Australian BTV-1 collected in the Northern Territory in 1979. Two distinct profiles were found. Northern Territory isolates exhibited the prototype profile, yet those from Queensland had a significantly different ('resistant') profile. Nucleotide sequencing of gene segment 2 from both groups of isolates was undertaken. When the nucleotide sequences of isolates from a later period in each State were analysed (1997-2001), all exhibited the 'resistant' profile. Thus, a novel VP2 phenotype, already in existence in Queensland, had supplanted a pre-existing VP2 phenotype in the Northern Territory between the two periods. Furthermore, amino acid differences between the resistant and prototype VP2 proteins were analogous to amino acid substitutions known to be associated with neutralisation resistance. The host immune response may therefore have contributed to selection of the 'resistant' phenotype.

Public health risks of the flesh of farmed crocodiles.

The farming of crocodiles in the Northern Territory of Australia is a rapidly growing industry. The saltwater crocodile produces a premium quality skin which is sought world-wide for the lucrative leather trade and manufacture of finished articles. Flesh is considered to be a by-product of skin production. Several procedures are used in abattoirs to prevent the risk of cross contamination of flesh. The public health risks linked to the production of crocodile flesh are described for the two main diseases of concern, namely: sparganosis and salmonellosis. The slaughter and hygienic processing procedures and local laboratory evidence indicate that the consumption of crocodile flesh produced in the Northern Territory carries a negligible public health risk.

Recall of family factors in social phobia and panic disorder: comparison of mother and offspring reports.

Previous research has indicated that adults with various anxiety disorders, especially social phobia, recall their parents as excessively protective and controlling and as low in socialization. However, it is not clear whether such results would be supported by parents. In the present study subjects with social phobia, panic disorder, and nonclinical subjects and their mothers were given parallel measures of maternal control, socialization, and offspring early introverted behaviors as well as several questions relating to two early major life events and family size. Anxious offspring reported the usual high maternal control and low paternal socialization and mother supported the data on socialization. On control, mothers provided mixed results, disagreeing on a more standard measure, but showing agreement on a more operationalized measure. The data were more consistent for social phobia than for panic disorder. In terms of early life factors, both anxiety disorders were associated with fewer friends and more introverted behaviors, while family size and two major life events did not differentiate groups.

EHDV-1, a new Australian serotype of epizootic haemorrhagic disease virus isolated from sentinel cattle in the Northern Territory.

In 1992, a virus (DPP2209) isolated from sentinel cattle located at Coastal Plains Research Station, latitude 12 degrees 39'S, longitude 131 degrees 20'E, approximately 60 km east of Darwin, Northern Territory. This virus was identified as a serotype of epizootic haemorrhagic disease (EHD) of deer virus previously undescribed in Australia. An additional 17 isolation of this virus were made from eight animals during the period February to May. Electron microscopic studies showed the presence of orbivirus-like structures. Serogrouping ELISA, indirect immunofluorescence assay and the serogrouping plaque reduction neutralisation test indicated the virus was a member of the epizootic haemorrhagic disease serogroup. Serotype specific plaque reduction neutralisation tests, indicated the virus was a member of the epizootic haemorrhagic disease serogroup not previously isolated in Australia. Analysis of the VP3 gene confirmed this observation. Cross neutralisation testing of the isolate with known epizootic haemorrhagic disease serotype viruses including endemic Australian and exotic strains identified isolate DPP2209 as epizootic haemorrhagic disease virus serotype 1.

Memory bias in social phobia.

To examine memory bias for social threat in social phobics, four studies are reported in which social phobic and nonclinical Ss are compared on their retrieval of threat-relevant information. Study 1 measured standard recall and recognition of threat, neutral, and positive words, while Study 2 assessed retrieval of these words through implicit and explicit tasks. The two final studies attempted to increase the validity of the procedures. Study 3 examined recall of feedback in a hypothetical public performance task and Study 4 examined autobiographical memory for social and neutral situations. All four studies consistently failed to find any evidence for biased retrieval processes in social phobics.

Koolpinyah: a virus related to kotonkan from cattle in northern Australia.

Two closely related viruses were isolated from the blood of bovines near Darwin, Northern Territory, Australia. When studies of virus morphology indicated that these were rhabdoviruses, serologic studies were done. These isolates are closely related or identical and are related to, but distinct from, the rabies-related kotonkan virus. Other serologic studies showed that these are two isolates of a newly recognized virus, for which the name Koolpinyah virus is proposed.

Investigations of bluetongue and other arboviruses in the blood and semen of naturally infected bulls.

Small groups of bulls were exposed to natural infection with arboviruses. The bulls were bled and ejaculated regularly and the blood and semen were processed for virus isolation. Over a 5-year observation period, virus isolation and serology indicated that the 29 exposed bulls had experienced 79 viraemic episodes with the viruses of the bluetongue, epizootic haemorrhagic disease, Palyam and Simbu serogroups and an incompletely characterised rhabdovirus. In no instance was there unequivocal evidence of bluetongue virus contamination of semen, despite 18 infections in the study period.

Arboviruses recovered from sentinel livestock in northern Australia.

Over 700 arboviruses were recovered between 1981 and 1987 from the blood of sentinel livestock near Darwin. Twenty-three isolates were made from sheep, goats, swamp buffalo (Bubalus bubalis) and horses, and the remainder were from cattle. The isolates have been typed as 27 separate viruses belonging to the bluetongue, epizootic haemorrhagic disease, Palyam, Simbu, bovine ephemeral fever, Tibrogargan and alphavirus groups. Ten of these viruses have not been isolated elsewhere in Australia and four have been isolated only in Darwin. Considerable annual variations in virus activity and in the durations of detectable viraemia were observed.