To compare reproduction in extensively managed, tropically adapted beef cows that were either ser... more To compare reproduction in extensively managed, tropically adapted beef cows that were either seropositive or seronegative to Neospora caninum. Longitudinal study of cows within management groups. Compare pregnancy with weaning outcomes for 502 seropositive and 3255 seronegative cows in 25 management groups. We found N. caninum in all herds, with an average of 20% of 2640 tested animals seropositive within management group; prevalence varied between 0% and 94%. At 7 of 10 sites assessed, there was evidence of horizontal transmission of N. caninum. There was no overall difference in pregnancy rate (79% vs 75%; P > 0.05), reproductive wastage after confirmed pregnancy diagnosis (11% vs 10%; P > 0.05) or weaning rate (67% vs 68%; P > 0.05) between seronegative and seropositive cows, respectively. In one herd where a combination of risk factors for N. caninum was present, a significant reduction in pregnancy rate occurred after the 6 months mating (85% vs 69%; P < 0.05). The fetal and calf losses observed were lowest in south-east Queensland (4.3% of 117 pregnancies), highest in north-west Queensland (15.5% of 413 pregnancies) and intermediate in north-east Queensland (10.2% of 1625 pregnancies). Other infectious agents that are known to cause reproductive wastage were endemic in many herds, though none appeared to cause significant fetal or calf loss in this study. Despite a high prevalence of N. caninum, there was no apparent effect on beef cattle reproduction, but there is potential to cause reproductive wastage if known risk factors to neosporosis are in effect.
Hendra virus occasionally causes severe disease in horses and humans. In Australia in 2013, infec... more Hendra virus occasionally causes severe disease in horses and humans. In Australia in 2013, infection was detected in a dog that had been in contact with an infected horse. Abnormalities and viral RNA were found in the dog's kidney, brain, lymph nodes, spleen, and liver. Dogs should be kept away from infected horses.
Bungowannah virus is the most divergent pestivirus, and both origin and reservoir host have not b... more Bungowannah virus is the most divergent pestivirus, and both origin and reservoir host have not been identified so far. We therefore performed in vitro tropism studies, which showed that Bungowannah virus differs remarkably from other pestiviruses. Interestingly, cell lines of vervet monkey, mouse, human and even of bat origin were susceptible. This broad in vitro tropism was not observed for a chimeric bovine viral diarrhoea virus (BVDV) expressing all structural proteins of Bungowannah virus. The viral envelope was not sufficient to completely transfer the cell tropism of Bungowannah virus to another pestivirus, and viral RNA replication was either markedly reduced or not detectable in a number of different cell lines for the tested BVDV strain and the chimera. We therefore suggest that the replication machinery together with the viral envelope is responsible for the unique broad cell tropism of Bungowannah virus.
Viruses belonging to the species Wallal virus and Warrego virus of the genus Orbivirus were ident... more Viruses belonging to the species Wallal virus and Warrego virus of the genus Orbivirus were identified as causative agents of blindness in marsupials in Australia during 1994/5. Recent comparisons of nucleotide (nt) and amino acid (aa) sequences have provided a basis for the grouping and classification of orbivirus isolates. However, full-genome sequence data are not available for representatives of all Orbivirus species. We report full-genome sequence data for three additional orbiviruses: Wallal virus (WALV); Mudjinabarry virus (MUDV) and Warrego virus (WARV). Comparisons of conserved polymerase (Pol), sub-core-shell 'T2' and core-surface 'T13' proteins show that these viruses group with other Culicoides borne orbiviruses, clustering with Eubenangee virus (EUBV), another orbivirus infecting marsupials. WARV shares ,70% aa identity in all three conserved proteins (Pol, T2 and T13) with other orbiviruses, consistent with its classification within a distinct Orbivirus species. Although WALV and MUDV share ,72.86%/67.93% aa/nt identity with other orbiviruses in Pol, T2 and T13, they share .99%/90% aa/nt identities with each other (consistent with membership of the same virus species -Wallal virus). However, WALV and MUDV share ,68% aa identity in their larger outer capsid protein VP2(OC1), consistent with membership of different serotypes within the species -WALV-1 and WALV-2 respectively.
In 2003 an outbreak of sudden deaths occurred in 3-4-week-old piglets on a farm in New South Wale... more In 2003 an outbreak of sudden deaths occurred in 3-4-week-old piglets on a farm in New South Wales, Australia. There was a marked increase in the birth of stillborn foetuses. Pathological changes consisted of a multifocal non-suppurative myocarditis. A viral infection was suspected but a wide range of known agents were excluded. A modified sequence independent single primer amplification (SISPA) method was used to identify a novel virus associated with this outbreak. Conserved 5 UTR motifs, the presence of a putative N pro coding region and limited antigenic cross-reactivity with other members of the Pestivirus genus, support the placement of this virus in the Pestivirus genus. Phylogenetic analysis of the 5 UTR, N pro and E2 coding regions showed this virus to be the most divergent pestivirus identified to date.
In recent years several atypical pestiviruses have been described. Bungowannah virus is the most ... more In recent years several atypical pestiviruses have been described. Bungowannah virus is the most divergent virus in this group. Therefore, heterologous complementation was used to clarify the phylogenetic relationship and to analyze the exchangeability of genome regions encoding structural proteins. Using a BVDV type 1 backbone, chimeric constructs with substituted envelope proteins E(rns), E1 and E2, were investigated. While all constructs replicated autonomously, infectious high titer chimeric virus could only be observed after exchanging the complete E1-E2 encoding region. The complementation of E1 and E2 alone resulted only in replicons. Complementation of BVDV-E(rns) was only efficient if Bungowannah virus-E(rns) was expressed from a bicistronic construct. Our data provide new insights in the compatibility of pestivirus proteins and demonstrate that heterologous complementation could be useful to characterize new pestiviruses.
Bungowannah virus is a pestivirus identified from an outbreak of stillbirth and increased mortali... more Bungowannah virus is a pestivirus identified from an outbreak of stillbirth and increased mortality in the first 3-4 weeks of life on a piggery in New South Wales, Australia in June 2003. The aims of this study were to determine if post-natal infection results in any clinical abnormalities and quantify the amount of Bungowannah virus RNA in blood, oropharyngeal, nasal and conjunctival excretions and faeces during the course of infection. Thirty pigs were infected intra-nasally with one of six different doses of Bungowannah virus or a control inoculum and clinical signs and rectal temperatures monitored. Sera, leukocytes and oropharyngeal, nasal, conjunctival, rectal and tissue swabs were tested for Bungowannah virus by qRT-PCR and sera for antibody by peroxidase linked assay and virus neutralisation test. The infectious dose by the intra-nasal route in weaner pigs was determined to be between 1.6 and 3.2 log(10) TCID(50). Few clinical signs could be attributed to infection. Viraemia and viral excretion in oropharyngeal secretions were detected from 3 days post-inoculation and seroconversion from 10 days post-inoculation. Viral shedding was greatest and most frequently detected in oropharyngeal, and to a lesser extent, nasal secretions, and generally detected in lower amounts and less frequently in conjunctival secretions and faeces.
In 2003 an outbreak of sudden deaths occurred in 2-3-week-old pigs on a piggery in New South Wale... more In 2003 an outbreak of sudden deaths occurred in 2-3-week-old pigs on a piggery in New South Wales, Australia. There was a marked increase in the birth of stillborn pigs and preweaning losses associated with a multifocal non-suppurative myocarditis with myonecrosis. The aim of this study was to amplify any infectious agents present in field material to aid the detection and identification of the causative agent of the porcine myocarditis syndrome (PMC). Foetuses were directly inoculated in utero with tissue extracts from field cases of PMC at 56-60, 70-84 or 85-94 days of gestation and euthanased 7-28 days later. The IgG concentration in foetal sera/body fluids was measured, hearts were examined by light microscopy and selected hearts were examined by electron microscopy. An infectious agent was detected in tissues from cases of PMC and its identification as the novel pestivirus Bungowannah virus has recently been reported (Kirkland et al., 2007). Sow sera, foetal tissues and foetal sera/body fluids were tested for Bungowannah virus RNA by qRT-PCR and antibody by peroxidase-linked assay. Bungowannah virus was detected in numerous organs of the porcine foetus. Following direct foetal exposure it is probable that this virus spreads by direct intra-uterine transmission to adjacent foetuses and by trans-uterine transmission to the dam. Data were obtained for both the replication of the virus in the porcine foetus and the humoral immune response in the foetus and sow.
In 2003 an outbreak of sudden deaths occurred in 2-3-week-old piglets on a piggery in New South W... more In 2003 an outbreak of sudden deaths occurred in 2-3-week-old piglets on a piggery in New South Wales, Australia. There was a marked increase in the birth of stillborn piglets and preweaning losses associated with a multifocal non-suppurative myocarditis with myonecrosis. The aim of this study was to review existing data and to undertake further investigations of specimens from naturally infected pigs to provide evidence to support the hypothesis that Bungowannah virus, a recently recognised pestivirus, causes the porcine myocarditis syndrome (PMC). Sera collected from gilts and sows from affected and unaffected units were tested for Bungowannah virus antibody by a peroxidase-linked assay and Bungowannah virus RNA by qRT-PCR in selected cases. Stillborn piglets from affected and an unaffected unit were also tested for Bungowannah virus antibody and RNA. Body fluid IgG levels and the incidence of myocardial lesions in these stillborn piglets are summarised. Tissue sections from stillborn piglets with myocarditis/myonecrosis were examined for Bungowannah virus RNA by in situ hybridisation. A clear temporal association between the occurrence of PMC on a unit or module and exposure to Bungowannah virus was identified by serological tests in both breeding aged animals and stillborn pigs. In addition, at the individual animal level on affected units, Bungowannah virus RNA was detected in stillborn piglets in large amounts by qRT-PCR and in association with myocardial lesions by in situ hybridisation. The examination of field material from cases of PMC by serology, qRT-PCR and in situ hybridisation provides strong indirect evidence that Bungowannah virus is the causative agent for PMC.
New Zealand is free from equine influenza and has never experienced an incursion in its horse pop... more New Zealand is free from equine influenza and has never experienced an incursion in its horse population. As part of New Zealand's preparedness to an incursion of an exotic animal disease, it was considered necessary to select the most accurate test for equine influenza (EI) from the array of those available. Four readily available blocking/ competitive enzyme-linked immunosorbent assays (ELISA), originally developed and marketed for the detection of antibodies against the avian influenza virus, were evaluated using serum samples from New Zealand non-infected, non-vaccinated horses (n = 365), and Australian field infected (n = 99) and experimentally infected horses (n = 3). Diagnostic specificities (DSP) and diagnostic sensitivities (DSE) were determined as follows: ELISA-1 = 98.1%/99.0%; ELISA-2 = 90.1%/99.0%; ELISA-3 = 98.1%/96.0%; ELISA-4 = 95.3%/99.0%. For ELISA-1, DSP and DSE results were comparable to previously published data on a larger sample number from Australian horses .
Eubenangee virus has previously been identified as the cause of Tammar sudden death syndrome (TSD... more Eubenangee virus has previously been identified as the cause of Tammar sudden death syndrome (TSDS). Eubenangee virus (EUBV), Tilligery virus (TILV), Pata virus (PATAV) and Ngoupe virus (NGOV) are currently all classified within the Eubenangee virus species of the genus Orbivirus, family Reoviridae. Full genome sequencing confirmed that EUBV and TILV (both of which are from Australia) show high levels of aa sequence identity (.92%) in the conserved polymerase VP1(Pol), sub-core VP3(T2) and outer core VP7(T13) proteins, and are therefore appropriately classified within the same virus species. However, they show much lower amino acid (aa) identity levels in their larger outer-capsid protein VP2 (,53%), consistent with membership of two different serotypes -EUBV-1 and EUBV-2 (respectively). In contrast PATAV showed significantly lower levels of aa sequence identity with either EUBV or TILV (with ,71% in VP1(Pol) and VP3(T2), and ,57% aa identity in VP7(T13)) consistent with membership of a distinct virus species. A proposal has therefore been sent to the Reoviridae Study Group of ICTV to recognise 'Pata virus' as a new Orbivirus species, with the PATAV isolate as serotype 1 (PATAV-1). Amongst the other orbiviruses, PATAV shows closest relationships to Epizootic Haemorrhagic Disease virus (EHDV), with 80.7%, 72.4% and 66.9% aa identity in VP3(T2), VP1(Pol), and VP7(T13) respectively. Although Ngoupe virus was not available for these studies, like PATAV it was isolated in Central Africa, and therefore seems likely to also belong to the new species, possibly as a distinct 'type'. The data presented will facilitate diagnostic assay design and the identification of additional isolates of these viruses.
Journal of Veterinary Diagnostic Investigation, 2010
Bovine viral diarrhea virus (BVDV) is divided into 2 different species within the Pestivirus genu... more Bovine viral diarrhea virus (BVDV) is divided into 2 different species within the Pestivirus genus, BVDV type 1 (BVDV-1) and BVDV type 2 (BVDV-2). Further phylogenetic analysis has revealed subgenotype groupings within the 2 types. Thus far, 12 BVDV-1 subgenotypes (a-l) and 2 BVDV-2 subgenotypes (a and b) have been identified. The purpose of the current study was to determine the prevalence of BVDV subgenotypes in the United States and Australia and to determine if there are detectable antigenic differences between the prevalent subgenotypes. To determine prevalence, phylogenetic analysis was performed on 2 blinded panels of isolates consisting of 351 viral isolates provided by the Elizabeth Macarthur Laboratory, New South Wales, and 514 viral isolates provided by Oklahoma State University. Differences were observed in the prevalence of BVDV subgenotypes between the United States (BVDV-1b most prevalent subgenotype) and Australia (BVDV-1c most prevalent subgenotype). To examine antigenic differences between the subgenotypes identified in samples from the United States and Australia, polyclonal antisera was produced in goats by exposing them at 3-week intervals to 2 noncytopathic and 1 cytopathic strain of either BVDV-1a, BVDV-1b, BVDV-1c, BVDV-2a, or Border disease virus (BDV). Virus neutralization (VN) assays were then performed against 3 viruses from each of the 5 subgenotypes. Comparison of VN results suggests that there are antigenic differences between BVDV strains belonging to different subgenotypes. The present study establishes a foundation for further studies examining whether vaccine protection can be improved by basing vaccines on the BVDV subgenotypes prevalent in the region in which the vaccine is to be used.
Journal of Veterinary Diagnostic Investigation, 2013
The New Zealand livestock population is free from Akabane virus (AKAV; family Bunyaviridae, genus... more The New Zealand livestock population is free from Akabane virus (AKAV; family Bunyaviridae, genus Orthobunyavirus), an arthropod-borne (arbo) virus, and the country is also free from the transmitting vector, biting midges of the genus Culicoides. 10,13 However, with the vector range and virus distribution in neighboring Australia and the evident global warming, incursion of vectors and viruses are a possibility. Therefore, an arbovirus surveillance program that includes AKAV surveillance has been operating in New Zealand since 1990, 11 which includes insect trapping and blood sampling from 17 sentinel cattle herds and testing with the AKAV neutralization test (VNT); to date with negative results. Laboratory diagnosis of Akabane disease is generally based on serology. The VNT is considered the reference method (gold standard) but is rather laborious and time consuming, requiring live virus and cell culture (Della-Porta AJ, White JR, Gard GP, Kirkland PD: 1993, Akabane diseasehistopathology, virology and serology. Available at: http:// www.scahls.org.au/__data/assets/pdf_file/0007/1280815/ akabane_diseases.pdf ). Commercial enzyme-linked immunosorbent assay (ELISA) kits are available, are easier to Abstract. In New Zealand, an arbovirus surveillance program has been operating for more than 20 years, which includes testing of cattle with the Akabane virus neutralization test. With the aim to replace this laborious test by an easier-to-perform enzyme-linked immunosorbent assay (ELISA), 2 commercial ELISA kits, ELISA-1 from France (originally from Australia) and ELISA-2 from Japan, were compared, using 334 serum samples from noninfected New Zealand cattle, and 548 serum samples from naturally infected cattle herds in Australia. Diagnostic specificities for the test methods were high, ranging from 99.4% to 100%. The diagnostic sensitivities varied considerably between the test methods and differed from the values reported by the manufacturers (94% for each ELISA). The diagnostic sensitivities relative to the virus neutralization test (n = 378) were 96.0% for ELISA-1 or 98.9% when suspect samples were included, and 78.0% for ELISA-2. Differences in the commercial ELISA kits may be explained by the presence of other Simbu serogroup viruses in Australian cattle herds, causing cross-reactions in ELISA-1. Both commercial ELISA kits would be fit for purpose and could replace the virus neutralization test for Akabane virus surveillance in New Zealand. ELISA-1 may be able to detect other Simbu serogroup viruses, should they be present. The current study shows that despite comparable ELISA test characteristics given by the manufacturers, evaluation on the target population revealed marked differences in the ELISA kits test methods' characteristics.
Background Development of effective and durable gene therapy for treatment of the respiratory man... more Background Development of effective and durable gene therapy for treatment of the respiratory manifestations of cystic fibrosis remains a formidable challenge. Obstacles include difficulty in achieving efficient gene transfer to mature airway epithelium and the need to stably transduce self-renewing epithelial progenitor cells in order to avoid loss of transgene expression through epithelial turnover. Targeting the developing airway epithelium during fetal life offers the prospect of circumventing these challenges.
After the introduction of pestivirus into a herd undergoing an embryo transfer and artificial ins... more After the introduction of pestivirus into a herd undergoing an embryo transfer and artificial insemination program, substantial post-weaning calf losses occurred. The predominant clinical feature was severe respiratory disease, in contrast to the commonly recognised mucosal disease. Thirty-one of 76 calves were affected, with a case fatality rate of 58%. All calves which were persistently infected with pestivirus died during the study period. There was a significant association in the surviving calves between the occurrence of recent pestivirus infection and respiratory disease. The losses on 'this property clearly indicate the need to routinely screen animals in an artificial breeding program for freedom from pestivirus infection.
Distributions of the vector Culicoides brevitarsis Kieffer (Diptera: Ceratopogonidae) (determined... more Distributions of the vector Culicoides brevitarsis Kieffer (Diptera: Ceratopogonidae) (determined from light trap data) and 2 arboviruses (determined from seroconversions in sentinel cattle) were studied in eastern New South Wales in 1993-94. C brevitarsis was recorded progressively from endemic areas on the north coast, to Nowra on the south coast, and westward to Scone, in the Hunter Valley. C brevitarsis also survived through winter at Paterson, in the Hunter Valley. Its apparently focal reappearance in this marginal area had no obvious effect on the broad pattern of its progression or the dispersal of Akabane and bluetongue viruses. These viruses were first recorded from foci near Coffs Harbour, on the mid-north coast. Their first occurrences at different locations were associated with those of C brevitarsis, but not with each other. The viruses were found only within the recorded limits of the vector's distribution. Delays between the initial occurrence of C brevitarsis and first evidence of virus transmissions at locations ranged from 2 to 7 months. The delays decreased away from the points of focus and were negatively associated with the time of initial occurrence of the vector. Seroconversions to the viruses were related to the presence of C brevitarsis. However, the densities of C brevitarsis had no apparent effect on the initial numbers of cattle seroconverting to either virus. The results support the conclusion that the progressions of C brevitarsis and Akabane and bluetongue viruses were the result of gradual movements by the vector.
To compare reproduction in extensively managed, tropically adapted beef cows that were either ser... more To compare reproduction in extensively managed, tropically adapted beef cows that were either seropositive or seronegative to Neospora caninum. Longitudinal study of cows within management groups. Compare pregnancy with weaning outcomes for 502 seropositive and 3255 seronegative cows in 25 management groups. We found N. caninum in all herds, with an average of 20% of 2640 tested animals seropositive within management group; prevalence varied between 0% and 94%. At 7 of 10 sites assessed, there was evidence of horizontal transmission of N. caninum. There was no overall difference in pregnancy rate (79% vs 75%; P > 0.05), reproductive wastage after confirmed pregnancy diagnosis (11% vs 10%; P > 0.05) or weaning rate (67% vs 68%; P > 0.05) between seronegative and seropositive cows, respectively. In one herd where a combination of risk factors for N. caninum was present, a significant reduction in pregnancy rate occurred after the 6 months mating (85% vs 69%; P < 0.05). The fetal and calf losses observed were lowest in south-east Queensland (4.3% of 117 pregnancies), highest in north-west Queensland (15.5% of 413 pregnancies) and intermediate in north-east Queensland (10.2% of 1625 pregnancies). Other infectious agents that are known to cause reproductive wastage were endemic in many herds, though none appeared to cause significant fetal or calf loss in this study. Despite a high prevalence of N. caninum, there was no apparent effect on beef cattle reproduction, but there is potential to cause reproductive wastage if known risk factors to neosporosis are in effect.
Hendra virus occasionally causes severe disease in horses and humans. In Australia in 2013, infec... more Hendra virus occasionally causes severe disease in horses and humans. In Australia in 2013, infection was detected in a dog that had been in contact with an infected horse. Abnormalities and viral RNA were found in the dog's kidney, brain, lymph nodes, spleen, and liver. Dogs should be kept away from infected horses.
Bungowannah virus is the most divergent pestivirus, and both origin and reservoir host have not b... more Bungowannah virus is the most divergent pestivirus, and both origin and reservoir host have not been identified so far. We therefore performed in vitro tropism studies, which showed that Bungowannah virus differs remarkably from other pestiviruses. Interestingly, cell lines of vervet monkey, mouse, human and even of bat origin were susceptible. This broad in vitro tropism was not observed for a chimeric bovine viral diarrhoea virus (BVDV) expressing all structural proteins of Bungowannah virus. The viral envelope was not sufficient to completely transfer the cell tropism of Bungowannah virus to another pestivirus, and viral RNA replication was either markedly reduced or not detectable in a number of different cell lines for the tested BVDV strain and the chimera. We therefore suggest that the replication machinery together with the viral envelope is responsible for the unique broad cell tropism of Bungowannah virus.
Viruses belonging to the species Wallal virus and Warrego virus of the genus Orbivirus were ident... more Viruses belonging to the species Wallal virus and Warrego virus of the genus Orbivirus were identified as causative agents of blindness in marsupials in Australia during 1994/5. Recent comparisons of nucleotide (nt) and amino acid (aa) sequences have provided a basis for the grouping and classification of orbivirus isolates. However, full-genome sequence data are not available for representatives of all Orbivirus species. We report full-genome sequence data for three additional orbiviruses: Wallal virus (WALV); Mudjinabarry virus (MUDV) and Warrego virus (WARV). Comparisons of conserved polymerase (Pol), sub-core-shell 'T2' and core-surface 'T13' proteins show that these viruses group with other Culicoides borne orbiviruses, clustering with Eubenangee virus (EUBV), another orbivirus infecting marsupials. WARV shares ,70% aa identity in all three conserved proteins (Pol, T2 and T13) with other orbiviruses, consistent with its classification within a distinct Orbivirus species. Although WALV and MUDV share ,72.86%/67.93% aa/nt identity with other orbiviruses in Pol, T2 and T13, they share .99%/90% aa/nt identities with each other (consistent with membership of the same virus species -Wallal virus). However, WALV and MUDV share ,68% aa identity in their larger outer capsid protein VP2(OC1), consistent with membership of different serotypes within the species -WALV-1 and WALV-2 respectively.
In 2003 an outbreak of sudden deaths occurred in 3-4-week-old piglets on a farm in New South Wale... more In 2003 an outbreak of sudden deaths occurred in 3-4-week-old piglets on a farm in New South Wales, Australia. There was a marked increase in the birth of stillborn foetuses. Pathological changes consisted of a multifocal non-suppurative myocarditis. A viral infection was suspected but a wide range of known agents were excluded. A modified sequence independent single primer amplification (SISPA) method was used to identify a novel virus associated with this outbreak. Conserved 5 UTR motifs, the presence of a putative N pro coding region and limited antigenic cross-reactivity with other members of the Pestivirus genus, support the placement of this virus in the Pestivirus genus. Phylogenetic analysis of the 5 UTR, N pro and E2 coding regions showed this virus to be the most divergent pestivirus identified to date.
In recent years several atypical pestiviruses have been described. Bungowannah virus is the most ... more In recent years several atypical pestiviruses have been described. Bungowannah virus is the most divergent virus in this group. Therefore, heterologous complementation was used to clarify the phylogenetic relationship and to analyze the exchangeability of genome regions encoding structural proteins. Using a BVDV type 1 backbone, chimeric constructs with substituted envelope proteins E(rns), E1 and E2, were investigated. While all constructs replicated autonomously, infectious high titer chimeric virus could only be observed after exchanging the complete E1-E2 encoding region. The complementation of E1 and E2 alone resulted only in replicons. Complementation of BVDV-E(rns) was only efficient if Bungowannah virus-E(rns) was expressed from a bicistronic construct. Our data provide new insights in the compatibility of pestivirus proteins and demonstrate that heterologous complementation could be useful to characterize new pestiviruses.
Bungowannah virus is a pestivirus identified from an outbreak of stillbirth and increased mortali... more Bungowannah virus is a pestivirus identified from an outbreak of stillbirth and increased mortality in the first 3-4 weeks of life on a piggery in New South Wales, Australia in June 2003. The aims of this study were to determine if post-natal infection results in any clinical abnormalities and quantify the amount of Bungowannah virus RNA in blood, oropharyngeal, nasal and conjunctival excretions and faeces during the course of infection. Thirty pigs were infected intra-nasally with one of six different doses of Bungowannah virus or a control inoculum and clinical signs and rectal temperatures monitored. Sera, leukocytes and oropharyngeal, nasal, conjunctival, rectal and tissue swabs were tested for Bungowannah virus by qRT-PCR and sera for antibody by peroxidase linked assay and virus neutralisation test. The infectious dose by the intra-nasal route in weaner pigs was determined to be between 1.6 and 3.2 log(10) TCID(50). Few clinical signs could be attributed to infection. Viraemia and viral excretion in oropharyngeal secretions were detected from 3 days post-inoculation and seroconversion from 10 days post-inoculation. Viral shedding was greatest and most frequently detected in oropharyngeal, and to a lesser extent, nasal secretions, and generally detected in lower amounts and less frequently in conjunctival secretions and faeces.
In 2003 an outbreak of sudden deaths occurred in 2-3-week-old pigs on a piggery in New South Wale... more In 2003 an outbreak of sudden deaths occurred in 2-3-week-old pigs on a piggery in New South Wales, Australia. There was a marked increase in the birth of stillborn pigs and preweaning losses associated with a multifocal non-suppurative myocarditis with myonecrosis. The aim of this study was to amplify any infectious agents present in field material to aid the detection and identification of the causative agent of the porcine myocarditis syndrome (PMC). Foetuses were directly inoculated in utero with tissue extracts from field cases of PMC at 56-60, 70-84 or 85-94 days of gestation and euthanased 7-28 days later. The IgG concentration in foetal sera/body fluids was measured, hearts were examined by light microscopy and selected hearts were examined by electron microscopy. An infectious agent was detected in tissues from cases of PMC and its identification as the novel pestivirus Bungowannah virus has recently been reported (Kirkland et al., 2007). Sow sera, foetal tissues and foetal sera/body fluids were tested for Bungowannah virus RNA by qRT-PCR and antibody by peroxidase-linked assay. Bungowannah virus was detected in numerous organs of the porcine foetus. Following direct foetal exposure it is probable that this virus spreads by direct intra-uterine transmission to adjacent foetuses and by trans-uterine transmission to the dam. Data were obtained for both the replication of the virus in the porcine foetus and the humoral immune response in the foetus and sow.
In 2003 an outbreak of sudden deaths occurred in 2-3-week-old piglets on a piggery in New South W... more In 2003 an outbreak of sudden deaths occurred in 2-3-week-old piglets on a piggery in New South Wales, Australia. There was a marked increase in the birth of stillborn piglets and preweaning losses associated with a multifocal non-suppurative myocarditis with myonecrosis. The aim of this study was to review existing data and to undertake further investigations of specimens from naturally infected pigs to provide evidence to support the hypothesis that Bungowannah virus, a recently recognised pestivirus, causes the porcine myocarditis syndrome (PMC). Sera collected from gilts and sows from affected and unaffected units were tested for Bungowannah virus antibody by a peroxidase-linked assay and Bungowannah virus RNA by qRT-PCR in selected cases. Stillborn piglets from affected and an unaffected unit were also tested for Bungowannah virus antibody and RNA. Body fluid IgG levels and the incidence of myocardial lesions in these stillborn piglets are summarised. Tissue sections from stillborn piglets with myocarditis/myonecrosis were examined for Bungowannah virus RNA by in situ hybridisation. A clear temporal association between the occurrence of PMC on a unit or module and exposure to Bungowannah virus was identified by serological tests in both breeding aged animals and stillborn pigs. In addition, at the individual animal level on affected units, Bungowannah virus RNA was detected in stillborn piglets in large amounts by qRT-PCR and in association with myocardial lesions by in situ hybridisation. The examination of field material from cases of PMC by serology, qRT-PCR and in situ hybridisation provides strong indirect evidence that Bungowannah virus is the causative agent for PMC.
New Zealand is free from equine influenza and has never experienced an incursion in its horse pop... more New Zealand is free from equine influenza and has never experienced an incursion in its horse population. As part of New Zealand's preparedness to an incursion of an exotic animal disease, it was considered necessary to select the most accurate test for equine influenza (EI) from the array of those available. Four readily available blocking/ competitive enzyme-linked immunosorbent assays (ELISA), originally developed and marketed for the detection of antibodies against the avian influenza virus, were evaluated using serum samples from New Zealand non-infected, non-vaccinated horses (n = 365), and Australian field infected (n = 99) and experimentally infected horses (n = 3). Diagnostic specificities (DSP) and diagnostic sensitivities (DSE) were determined as follows: ELISA-1 = 98.1%/99.0%; ELISA-2 = 90.1%/99.0%; ELISA-3 = 98.1%/96.0%; ELISA-4 = 95.3%/99.0%. For ELISA-1, DSP and DSE results were comparable to previously published data on a larger sample number from Australian horses .
Eubenangee virus has previously been identified as the cause of Tammar sudden death syndrome (TSD... more Eubenangee virus has previously been identified as the cause of Tammar sudden death syndrome (TSDS). Eubenangee virus (EUBV), Tilligery virus (TILV), Pata virus (PATAV) and Ngoupe virus (NGOV) are currently all classified within the Eubenangee virus species of the genus Orbivirus, family Reoviridae. Full genome sequencing confirmed that EUBV and TILV (both of which are from Australia) show high levels of aa sequence identity (.92%) in the conserved polymerase VP1(Pol), sub-core VP3(T2) and outer core VP7(T13) proteins, and are therefore appropriately classified within the same virus species. However, they show much lower amino acid (aa) identity levels in their larger outer-capsid protein VP2 (,53%), consistent with membership of two different serotypes -EUBV-1 and EUBV-2 (respectively). In contrast PATAV showed significantly lower levels of aa sequence identity with either EUBV or TILV (with ,71% in VP1(Pol) and VP3(T2), and ,57% aa identity in VP7(T13)) consistent with membership of a distinct virus species. A proposal has therefore been sent to the Reoviridae Study Group of ICTV to recognise 'Pata virus' as a new Orbivirus species, with the PATAV isolate as serotype 1 (PATAV-1). Amongst the other orbiviruses, PATAV shows closest relationships to Epizootic Haemorrhagic Disease virus (EHDV), with 80.7%, 72.4% and 66.9% aa identity in VP3(T2), VP1(Pol), and VP7(T13) respectively. Although Ngoupe virus was not available for these studies, like PATAV it was isolated in Central Africa, and therefore seems likely to also belong to the new species, possibly as a distinct 'type'. The data presented will facilitate diagnostic assay design and the identification of additional isolates of these viruses.
Journal of Veterinary Diagnostic Investigation, 2010
Bovine viral diarrhea virus (BVDV) is divided into 2 different species within the Pestivirus genu... more Bovine viral diarrhea virus (BVDV) is divided into 2 different species within the Pestivirus genus, BVDV type 1 (BVDV-1) and BVDV type 2 (BVDV-2). Further phylogenetic analysis has revealed subgenotype groupings within the 2 types. Thus far, 12 BVDV-1 subgenotypes (a-l) and 2 BVDV-2 subgenotypes (a and b) have been identified. The purpose of the current study was to determine the prevalence of BVDV subgenotypes in the United States and Australia and to determine if there are detectable antigenic differences between the prevalent subgenotypes. To determine prevalence, phylogenetic analysis was performed on 2 blinded panels of isolates consisting of 351 viral isolates provided by the Elizabeth Macarthur Laboratory, New South Wales, and 514 viral isolates provided by Oklahoma State University. Differences were observed in the prevalence of BVDV subgenotypes between the United States (BVDV-1b most prevalent subgenotype) and Australia (BVDV-1c most prevalent subgenotype). To examine antigenic differences between the subgenotypes identified in samples from the United States and Australia, polyclonal antisera was produced in goats by exposing them at 3-week intervals to 2 noncytopathic and 1 cytopathic strain of either BVDV-1a, BVDV-1b, BVDV-1c, BVDV-2a, or Border disease virus (BDV). Virus neutralization (VN) assays were then performed against 3 viruses from each of the 5 subgenotypes. Comparison of VN results suggests that there are antigenic differences between BVDV strains belonging to different subgenotypes. The present study establishes a foundation for further studies examining whether vaccine protection can be improved by basing vaccines on the BVDV subgenotypes prevalent in the region in which the vaccine is to be used.
Journal of Veterinary Diagnostic Investigation, 2013
The New Zealand livestock population is free from Akabane virus (AKAV; family Bunyaviridae, genus... more The New Zealand livestock population is free from Akabane virus (AKAV; family Bunyaviridae, genus Orthobunyavirus), an arthropod-borne (arbo) virus, and the country is also free from the transmitting vector, biting midges of the genus Culicoides. 10,13 However, with the vector range and virus distribution in neighboring Australia and the evident global warming, incursion of vectors and viruses are a possibility. Therefore, an arbovirus surveillance program that includes AKAV surveillance has been operating in New Zealand since 1990, 11 which includes insect trapping and blood sampling from 17 sentinel cattle herds and testing with the AKAV neutralization test (VNT); to date with negative results. Laboratory diagnosis of Akabane disease is generally based on serology. The VNT is considered the reference method (gold standard) but is rather laborious and time consuming, requiring live virus and cell culture (Della-Porta AJ, White JR, Gard GP, Kirkland PD: 1993, Akabane diseasehistopathology, virology and serology. Available at: http:// www.scahls.org.au/__data/assets/pdf_file/0007/1280815/ akabane_diseases.pdf ). Commercial enzyme-linked immunosorbent assay (ELISA) kits are available, are easier to Abstract. In New Zealand, an arbovirus surveillance program has been operating for more than 20 years, which includes testing of cattle with the Akabane virus neutralization test. With the aim to replace this laborious test by an easier-to-perform enzyme-linked immunosorbent assay (ELISA), 2 commercial ELISA kits, ELISA-1 from France (originally from Australia) and ELISA-2 from Japan, were compared, using 334 serum samples from noninfected New Zealand cattle, and 548 serum samples from naturally infected cattle herds in Australia. Diagnostic specificities for the test methods were high, ranging from 99.4% to 100%. The diagnostic sensitivities varied considerably between the test methods and differed from the values reported by the manufacturers (94% for each ELISA). The diagnostic sensitivities relative to the virus neutralization test (n = 378) were 96.0% for ELISA-1 or 98.9% when suspect samples were included, and 78.0% for ELISA-2. Differences in the commercial ELISA kits may be explained by the presence of other Simbu serogroup viruses in Australian cattle herds, causing cross-reactions in ELISA-1. Both commercial ELISA kits would be fit for purpose and could replace the virus neutralization test for Akabane virus surveillance in New Zealand. ELISA-1 may be able to detect other Simbu serogroup viruses, should they be present. The current study shows that despite comparable ELISA test characteristics given by the manufacturers, evaluation on the target population revealed marked differences in the ELISA kits test methods' characteristics.
Background Development of effective and durable gene therapy for treatment of the respiratory man... more Background Development of effective and durable gene therapy for treatment of the respiratory manifestations of cystic fibrosis remains a formidable challenge. Obstacles include difficulty in achieving efficient gene transfer to mature airway epithelium and the need to stably transduce self-renewing epithelial progenitor cells in order to avoid loss of transgene expression through epithelial turnover. Targeting the developing airway epithelium during fetal life offers the prospect of circumventing these challenges.
After the introduction of pestivirus into a herd undergoing an embryo transfer and artificial ins... more After the introduction of pestivirus into a herd undergoing an embryo transfer and artificial insemination program, substantial post-weaning calf losses occurred. The predominant clinical feature was severe respiratory disease, in contrast to the commonly recognised mucosal disease. Thirty-one of 76 calves were affected, with a case fatality rate of 58%. All calves which were persistently infected with pestivirus died during the study period. There was a significant association in the surviving calves between the occurrence of recent pestivirus infection and respiratory disease. The losses on 'this property clearly indicate the need to routinely screen animals in an artificial breeding program for freedom from pestivirus infection.
Distributions of the vector Culicoides brevitarsis Kieffer (Diptera: Ceratopogonidae) (determined... more Distributions of the vector Culicoides brevitarsis Kieffer (Diptera: Ceratopogonidae) (determined from light trap data) and 2 arboviruses (determined from seroconversions in sentinel cattle) were studied in eastern New South Wales in 1993-94. C brevitarsis was recorded progressively from endemic areas on the north coast, to Nowra on the south coast, and westward to Scone, in the Hunter Valley. C brevitarsis also survived through winter at Paterson, in the Hunter Valley. Its apparently focal reappearance in this marginal area had no obvious effect on the broad pattern of its progression or the dispersal of Akabane and bluetongue viruses. These viruses were first recorded from foci near Coffs Harbour, on the mid-north coast. Their first occurrences at different locations were associated with those of C brevitarsis, but not with each other. The viruses were found only within the recorded limits of the vector's distribution. Delays between the initial occurrence of C brevitarsis and first evidence of virus transmissions at locations ranged from 2 to 7 months. The delays decreased away from the points of focus and were negatively associated with the time of initial occurrence of the vector. Seroconversions to the viruses were related to the presence of C brevitarsis. However, the densities of C brevitarsis had no apparent effect on the initial numbers of cattle seroconverting to either virus. The results support the conclusion that the progressions of C brevitarsis and Akabane and bluetongue viruses were the result of gradual movements by the vector.
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