Use of spatio-temporal habitat suitability modelling to prioritise areas for common carp biocontrol in Australia using the virus CyHV-3.

Common carp (Cyprinus carpio) are an invasive species of the rivers and waterways of south-eastern Australia, implicated in the serious decline of many native fish species. Over the past 50 years a variety of control options have been explored, all of which to date have proved either ineffective or cost prohibitive. Most recently the use of cyprinid herpesvirus 3 (CyHV-3) has been proposed as a biocontrol agent, but to assess the risks and benefits of this, as well as to develop a strategy for the release of the virus, a knowledge of the fundamental processes driving carp distribution and abundance is required. To this end, we developed a novel process-based modelling framework that integrates expert opinion with spatio-temporal datasets via the construction of a Bayesian Network. The resulting weekly networks thus enabled an estimate of the habitat suitability for carp across a range of hydrological habitats in south-eastern Australia, covering five diverse catchment areas encompassing in total a drainage area of 132,129 km2 over a period of 17-27 years. This showed that while suitability for adult and subadult carp was medium-high across most habitats throughout the period, nevertheless the majority of habitats were poorly suited for the recruitment of larvae and young-of-year (YOY). Instead, high population abundance was confirmed to depend on a small number of recruitment hotspots which occur in years of favourable inundation. Quantification of the underlying ecological drivers of carp abundance thus makes possible detailed planning by focusing on critical weaknesses in the population biology of carp. More specifically, it permits the rational planning for population reduction using the biocontrol agent, CyHV-3, targeting areas where the total population density is above a "damage threshold" of approximately 100 kg/ha.

Cyprinid herpesvirus 3 as a potential biological control agent for carp (Cyprinus carpio) in Australia: susceptibility of non-target species.

Carp (Cyprinus carpio L.) is a pest species in Australian waterways, and cyprinid herpesvirus 3 (CyHV-3) is being considered as a potential biological control (biocontrol) agent. An important consideration for any such agent is its target specificity. In this study, the susceptibility to CyHV-3 of a range of non-target species (NTS) was tested. The NTS were as follows: 13 native Australian, and one introduced, fish species; a lamprey species; a crustacean; two native amphibian species (tadpole and mature stages); two native reptilian species; chickens; and laboratory mice. Animals were exposed to 100-1000 times the approximate minimum amount of CyHV-3 required to cause disease in carp by intraperitoneal and/or bath challenge, and then examined clinically each day over the course of 28 days post-challenge. There were no clinical signs, mortalities or histological evidence consistent with a viral infection in a wide taxonomic range of NTS. Furthermore, there was no molecular evidence of infection with CyHV-3, and, in particular, all RT-PCRs for viral mRNA were negative. As a consequence, the results encourage further investigation of CyHV-3 as a potential biocontrol agent that is specific for carp.

Development and application of molecular methods (PCR) for detection of Tasmanian Atlantic salmon reovirus.

Molecular (PCR) diagnostic tests for the detection and identification of aquareovirus in general, and Tasmanian Atlantic salmon reovirus (TSRV) specifically, were developed, and their diagnostic sensitivity and specificity were determined and compared with virus isolation in cell culture. Intralaboratory and interlaboratory comparison of PCR (conventional hemi-nested RT-PCR & RT-qPCR) and virus isolation in cell culture using finfish cell lines, CHSE-214 and EPC, was carried out for the detection and identification of TSRV using field samples of farmed Atlantic salmon Salmo salar, L. from various aquaculture sites around Tasmania. The interlaboratory comparison of diagnostic methods was carried out between two laboratories, AAHL-CSIRO and DPIPWE-Tasmania. A total of 144 fish from nine sites (12-33 fish per site) were sampled from two regions of Tasmania (Tamar River estuary in the north and Huon River estuary in the south-east) during late spring to early summer of 2009, and the data were analysed using different statistical approaches. The prevalence of TSRV ranged from 6% to 22% in both regions. All the diagnostic methods (data from both laboratories) had high specificity, while the estimated sensitivity varied between tests with RT-qPCR being the most sensitive (95.2%) method followed by virus isolation and then conventional hemi-nested RT-PCR.

The spatial and temporal variation of the distribution and prevalence of Atlantic salmon reovirus (TSRV) infection in Tasmania, Australia.

Atlantic salmon reovirus (TSRV) has been consistently isolated from Atlantic salmon in Tasmania, since first identification in 1990 under the Tasmanian Salmonid Health Surveillance Program (TSHSP). The distribution and prevalence of TSRV was identified using TSHSP data. A data set of 730 fish submissions tested over a period of 15 years was reviewed and analysed to describe the spatial and temporal variation of TSRV in Tasmanian salmonid aquaculture production units. The virus was present throughout Tasmania with the highest reported prevalence of the virus in the south-east region of Tasmania.

Role of genetically engineered animals in future food production.

Genetically engineered (GE) animals are likely to have an important role in the future in meeting the food demand of a burgeoning global population. There have already been many notable achievements using this technology in livestock, poultry and aquatic species. In particular, the use of RNA interference (RNAi) to produce virus-resistant animals is a rapidly-developing area of research. However, despite the promise of this technology, very few GE animals have been commercialised. This review aims to provide information so that veterinarians and animal health scientists are better able to participate in the debate on GE animals.

Isolation and characterization of koi herpesvirus (KHV) from Indonesia: identification of a new genetic lineage.

Koi herpesvirus (KHV) is the aetiological agent of an emerging disease (KHVD) associated with mass mortalities in koi and common carp and reported from at least 30 countries. We report the first isolation of KHV from koi and common carp in Indonesia and initial characterization of the isolates. Clinical signs, histopathology and virion morphology are similar to those of isolates from other countries. Phylogenetic analyses using the thymidine kinase gene amplified from each isolate and from carp tissue samples collected from KHVD outbreaks throughout Indonesia indicated that the Indonesian isolates are more closely related to the Asian than the European KHV lineage. Sequence analysis of two other variable regions between ORF29 and ORF31 (marker I) and near the start of ORF 133 (marker II) indicated that all Indonesian isolates displayed a marker I allele (I(++)) previously identified only in isolates of the Asian lineage. However, in the marker II region, all Indonesian isolates displayed the II(-) allele, which has been reported previously only amongst isolates of the European lineage, and nine of these displayed a mixed genotype (II(+)II(-)). The I(++)II(-) genotype has not been reported previously and appears to represent a new intermediate lineage that may have emerged in Indonesia.

Survey for the presence of White Spot Syndrome virus in Australian crustaceans.

Agriculture, Fisheries and Forestry - Australia, GPO Box 858, Canberra, Australian Capital Territory 2611.

Detection of White Spot Syndrome virus and Yellowhead virus in prawns imported into Australia.

OBJECTIVE: To determine whether viable White Spot Syndrome virus (WSSV) or Yellowhead virus (YHV) were present in prawn products imported into Australia. PROCEDURE: A sample of fourteen uncooked prawns was obtained from a consignment imported from southeast Asia. Each of the prawns was examined for WSSV by polymerase chain reaction (PCR), and then a bioassay was conducted in which a 10% homogenate of cuticular epithelium from each of the prawns was inoculated intramuscularly into healthy challenge prawns (Penaeus monodon) from Australia. The latter were then monitored for clinical signs of disease, and tissue samples were processed for electron microscopy, histological examination and for detection of WSSV by in situ hybridization (ISH) using a commercial kit. Limited numbers of haemolymph samples from inoculated challenge prawns were also examined by PCR for the presence of WSSV and YHV. All work was carried out under microbiologically secure conditions. RESULTS: Results of the initial PCR examination for WSSV on the imported prawns were not definitive. However, in the bioassay, several of the challenge prawns inoculated with homogenates from the imported prawns showed clinical signs of disease (inappetence and lethargy) within 24 h post inoculation (pi) and died at 1 to 4 days pi. Tissue samples from a number of moribund prawns demonstrated lesions typical of White Spot Disease (WSD), and the presence of the virus was confirmed by electron microscopy, ISH and PCR. YHV was also demonstrated by PCR in two challenge prawns inoculated with homogenates. CONCLUSION: Viable WSSV and YHV were present in frozen prawn products imported into Australia for human consumption from southeast Asia. Importation of frozen infected products may present a risk of transferring virus to wild and farmed populations of crustaceans in this country. To date, WSD and Yellowhead Disease remain exotic to Australia.

Pathogenesis studies with Australian bat lyssavirus in grey-headed flying foxes (Pteropus poliocephalus).

OBJECTIVE: To examine the susceptibility of the grey-headed flying fox (Pteropus poliocephalus) to Australian bat lyssavirus (ABL), and to provide preliminary observations on the pathogenesis of the disease in flying foxes. PROCEDURE: Ten flying foxes were inoculated intramuscularly with ABL, and four with a bat-associated rabies virus. Inoculated animals were observed daily, and clinical samples collected every 9 to 14 days. Animals with abnormal clinical signs were euthanased, and samples collected for histological, serological, virological and immunohistological examinations. At 3 months post inoculation (PI), all survivors were euthanased, and each submitted to a similar examination. RESULTS: Three ABL-inoculated flying foxes, and two rabies-inoculated animals developed abnormal clinical signs between 15 and 24 days PI. All three ABL-inoculated animals had histological lesions consistent with a lyssavirus infection, and lyssaviral antigen was identified in the central nervous system (CNS) of each. Virus was isolated from the brain of two affected animals. Of the rabies-inoculated flying foxes, both had histological lesions and viral antigen in the CNS. Virus was recovered from the brain of only one. None of the five affected flying foxes developed anti-lyssavirus antibodies, but, by 3 months PI, five of the seven ABL-inoculated survivors, and one of the two rabies virus-inoculated survivors, had seroconverted. The dynamics of the immune responses were quite variable. CONCLUSIONS: The response of flying foxes to ABL, administered by a peripheral route of inoculation, was similar to that of bats inoculated peripherally with bat-derived rabies viruses.

Evidence for insect transmission of rabbit haemorrhagic disease virus.

The spread of rabbit haemorrhagic disease (RHD) virus from quarantine on Wardang Island to mainland Australia in 1995 suggested that insects could be potential vectors. Field observations and laboratory experiments were conducted to address aspects of this hypothesis. Firstly, the variation in insect populations on the island during the field trials was examined. There was approximately a 1,000-fold increase in the number of bushflies, Musca vetustissima, shortly before the spread of the virus. Secondly, M. vetustissima were tested in the laboratory as potential vectors of RHD virus, and it was demonstrated that disease could be transmitted between rabbits by flies. Finally, 13 of 16 insect samples, collected from Wardang Island and from several sites on the mainland following the spread of virus off the island, were positive for the presence of RHD virus by a specific polymerase chain reaction (PCR). Only one sample contained sufficient infectious virus to kill a susceptible rabbit. These data, combined with previously published information on fly biology, suggested that flies, particularly bushflies, may be involved in the transmission of RHD virus. Other possible routes of spread were not assessed in this study.

The presence of cross-reactive antibodies to rabbit haemorrhagic disease virus in Australian wild rabbits prior to the escape of virus from quarantine.

Sera collected from Australian wild rabbits prior to the escape of rabbit haemorrhagic disease virus (RHDV) from Wardang Island were examined for RHDV antibodies using purified recombinant capsid protein VP60 expressed from baculovirus. A VP60-based indirect ELISA showed that 196 of 392 wild rabbit sera reacted (OD(450) >0.15) with VP60. Twenty sera (OD(450) ranging from 0.15-2.47), randomly chosen from the 196 positive sera, recognized the 64 kDa VP60 in Western blot analysis, indicating that the reactivity detected in ELISA is indeed specific to the VP60 antigen. In a separate study, sera of 23 rabbits from an RHD-free area after the escape of RHDV were tested by ELISA and 21 of the 23 rabbits were found to be positive. When these rabbits were challenged with a lethal dose of RHDV, 11 out of the 23 rabbits survived. The presence of RHDV-protective antibodies in some of these rabbits suggested that they had been exposed to a pre-existing non-virulent rabbit calicivirus closely related to RHDV. These results highlight the need to study the prevalence of, and to characterize, this viral agent in order to effectively control rabbit populations in Australia and New Zealand.

Bat lyssavirus infections.

Bats, which represent approximately 24% of all known mammalian species, frequently act as vectors of lyssaviruses. In particular, insectivorous bats play an important role in the epidemiology of rabies and some rabies-like viruses, while the haematophagous vampire bats are the major wildlife vector for rabies in Latin America. In contrast, the role of fruit bats (flying foxes) in the epidemiology of the recently discovered Australian bat lyssavirus is only just emerging. Information on the pathogenesis of lyssaviruses in bats is scarce. However, in general, mortality in bats infected via a natural route appears to be low, and seroconversion occurs in many of those that survive. While transmission of rabies from an infected bat may be via a bite, other routes are apparently also possible. Methods for the diagnosis of bat lyssavirus infections in bats and terrestrial mammals (including humans) are similar to the classical procedures for rabies. Measures for the prevention and control of these diseases are also similar to those for rabies, although additional innovative methods have been tested, specifically to control vampire bat rabies.

An outbreak of malignant catarrhal fever in young rusa deer (Cervus timorensis).

On the basis of clinical signs and histological findings eight 9-month-old male rusa deer (Cervus timorensis) were diagnosed with sheep associated-malignant catarrhal fever. Following a variable course involving rectal temperatures around 40.5 degrees C, depression, inappetence, diarrhoea, corneal opacity and hypopyon all animals died or were euthanased over a 5-week period. Severe multifocal vasculitis, mainly periglomerular and in the arcuate vessels were consistent histological findings which in the past have been adequate to confirm clinical diagnosis of sheep associated-malignant catarrhal fever. A nested polymerase chain reaction test has been used to detect a sheep associated-malignant catarrhal fever PRC product, 238 base-pairs in size, in DNA extracted from lymphocyte preparations. The result supported the diagnosis of sheep associated-malignant catarrhal fever in these deer.

Peripheral blood lymphocyte subsets in fleece rot-resistant and -susceptible sheep.

OBJECTIVE: To compare haematological values and lymphocyte phenotypes in the peripheral blood of fleece rot-resistant and -susceptible sheep. PROCEDURE: Experiments were conducted on 2- and 3-year-old Merino rams, flock 1 (17 rams) and flock 2 (32 rams), respectively. Within each flock, individual rams were classified as fleece rot-resistant or -susceptible, based on established criteria. Total and differential white cell counts, and indirect fluorescent antibody tests specific for B cells and T cells were performed on all sheep. The concentration of various subsets of circulating lymphocytes was then determined in each sheep. RESULTS: There were no significant differences between fleece rot-resistant and -susceptible sheep from either flock in the mean total or differential white cell counts. However, fleece rot-resistant rams in flock 1 did have a significantly higher concentration of circulating SBU-T1+ cells than fleece rot-susceptible rams from the same flock. No such difference was noted in the rams from flock 2. While all rams in flock 1 were free of clinical fleece rot, 24 rams in flock 2 (comprising all 17 fleece rot-susceptible and 7 of 15 fleece rot-resistant animals) had clinical signs of the disease. Fleece rot-free rams in this flock (irrespective of their classification as fleece rot-resistant or -susceptible) had significantly higher concentrations of circulating SBU-T1+ cells compared with fleece rot-affected animals. They also had significantly higher concentrations of circulating B cells, and total lymphocytes. CONCLUSIONS: An examination of peripheral blood lymphocyte subsets in fleece rot-resistant and -susceptible sheep revealed a possible association between resistance to fleece rot and the concentration of circulating SBU-T1+ cells.

Retrospective diagnosis of bluetongue virus in stored frozen and fixed tissue samples using PCR.

Stored frozen (-70 degrees C) and formalin-fixed tissue samples constitute a valuable resource for retrospective studies of infectious diseases, or for diagnostic investigations. The polymerase chain reaction (PCR) affords an accurate and rapid method for detection of viral nucleic acids. It was applied to stored tissue samples collected from sheep inoculated with two Australian serotypes of bluetongue virus, BTV 1 and 23, and two North American serotypes, BTV 11 and 17. Specific nested PCR products were detected in both frozen and formalin-fixed samples from the Australian sheep after storage for 3.5 years. The tissues from sheep inoculated with the North American serotypes yielded specific nested PCR products after storage at -70 degrees C for 14 years. No specific primary PCR products were detected in any frozen or formalin-fixed samples. The PCR assay offers a potential benefit for epidemiological studies, and for screening of stored semen, embryos and tissue banks.

The persistence of foot-and-mouth disease virus on wool.

Five Suffolk sheep, held in a high-security isolation room, were exposed for 2 hours to the aerosol of 3 mature pigs that had been infected with foot-and-mouth disease virus (FMDV), strain O1-BFS. The fleeces of 3 of the sheep were contaminated with FMDV at 2 days post exposure (dpe), while at 5 dpe the fleeces of all 5 sheep were more extensively, and more heavily, contaminated. The persistence of FMDV on contaminated wool was examined in vitro using multiple 0.5 g samples of Merino wool that were each contaminated with one of 3 strains of FMDV in tissue-cultured medium: O1-BFS, O-Morocco (O-MOR 9/91) or an Asia 1 strain (TAI 1/90). Wool samples were held at either 4 degrees C, 18 degrees C or 37 degrees C, and decay curves were established for each virus at each temperature. These curves predicted that O1-BFS, O-MOR 9/91 and TAI 1/90 would fall below detectable levels at 72, 70 and 48 days post contamination (pc), respectively, for wool stored at 4 degrees C; at 11, 12 and 12 days pc, respectively, for wool stored at 18 degrees C; and at 57, 68 and 33 hours pc, respectively, for wool stored at 37 degrees C. For wool contaminated with O1-BFS-infected sheep faeces, urine or blood, or with O1-BFS-infected cattle saliva, decay curves predicted virus to persist for 5 to 11 days pc at 18 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Phylogenetic characterisation of bluetongue viruses from naturally-infected insects, cattle and sheep in Australia.

The polymerase chain reaction was used to detect the presence of bluetongue virus (BTV) in a number of clinical and insect samples collected in the Northern Territory of Australia. Sequence analyses of the amplified BTV genes differentiated endemic Australian and exotic viruses. Two potential exotic BTV were detected as a result of PCR analyses of blood from sentinel animals and of the insect vector, Culicoides wadai. The detection of BTV in C wadai was the first direct demonstration of the presence of BTV in this potential vector. This new technology can significantly reduce the time taken for a diagnosis from a clinical sample and increase the amount of useful information obtained on a BTV isolate by using rapid sequencing techniques. Sequence data were used to differentiate between BTV20 isolated in 1975 and two isolates of the same serotype, isolated in 1992, and indicated that the latter were probably a recent incursion into Australia from Indonesia due to their greater VP3 sequence homology to the BTV9 (Java) than to Australian BTV isolates.

Bluetongue virus infection in sheep: haematological changes and detection by polymerase chain reaction.

Eight sheep vaccinated with 10(6) pfu of attenuated Australian bluetongue virus serotype 23 (BTV 23A) and eight BTV-free sheep were challenged with virulent BTV 23. There was little subsequent variation in the mean clinical score, or in the mean lymphocyte and platelet concentrations in the peripheral blood of the eight vaccinated sheep. There was a marked thrombocytopenia and lymphopenia in the naive sheep as the mean lymphocyte and platelet concentrations fell to a minimum at days 8 and 11 after inoculation, respectively. Similar changes were observed in three other naive sheep inoculated with field isolates of BTV 1,9 or 23. BTV was detected by nested polymerase chain reaction in whole blood of these sheep between days 6 and 28, in mononuclear leukocytes between days 3 and 14, and in platelets between days 6 and 21.

Cloning and nucleotide sequence determination of the major envelope glycoprotein (gp55) gene of hog cholera virus (Weybridge).

A 1.7 kb cDNA fragment corresponding to the coding region of the major envelope glycoprotein (gp55) of pestivirus hog cholera (Weybridge) was obtained using the polymerase chain reaction (PCR), and then cloned into pUC 8. The deduced amino acid sequence of gp55 showed a strong homology to that of HCV strains Brescia (94%) and Alfort (90%), and to a lesser extent to the closely related gp53 of bovine viral diarrhoea virus strain, NADL (65%). Eighteen cysteine residues were identified in the sequenced region, all of which were conserved between the gp55/gp53 sequences. This suggests that although the homology at the protein level may vary, there are strong conformational motifs which are conserved among the pestivirus envelope proteins.