Factors controlling volatile organic compounds in dwellings in Melbourne, Australia.

This study characterized indoor volatile organic compounds (VOCs) and investigated the effects of the dwelling characteristics, building materials, occupant activities, and environmental conditions on indoor VOC concentrations in 40 dwellings located in Melbourne, Australia, in 2008 and 2009. A total of 97 VOCs were identified. Nine VOCs, n-butane, 2-methylbutane, toluene, formaldehyde, acetaldehyde, d-limonene, ethanol, 2-propanol, and acetic acid, accounted for 68% of the sum of all VOCs. The median indoor concentrations of all VOCs were greater than those measured outdoors. The occupant density was positively associated with indoor VOC concentrations via occupant activities, including respiration and combustion. Terpenes were associated with the use of household cleaning and laundry products. A petroleum-like indoor VOC signature of alkanes and aromatics was associated with the proximity of major roads. The indoor VOC concentrations were negatively correlated (P < 0.05) with ventilation. Levels of VOCs in these Australian dwellings were lower than those from previous studies in North America and Europe, probably due to a combination of an ongoing temporal decrease in indoor VOC concentrations and the leakier nature of Australian dwellings.

Survival of hendra virus in the environment: modelling the effect of temperature.

Hendra virus (HeV), a highly pathogenic zoonotic paramyxovirus recently emerged from bats, is a major concern to the horse industry in Australia. Previous research has shown that higher temperatures led to lower virus survival rates in the laboratory. We develop a model of survival of HeV in the environment as influenced by temperature. We used 20 years of daily temperature at six locations spanning the geographic range of reported HeV incidents to simulate the temporal and spatial impacts of temperature on HeV survival. At any location, simulated virus survival was greater in winter than in summer, and in any month of the year, survival was higher in higher latitudes. At any location, year-to-year variation in virus survival 24 h post-excretion was substantial and was as large as the difference between locations. Survival was higher in microhabitats with lower than ambient temperature, and when environmental exposure was shorter. The within-year pattern of virus survival mirrored the cumulative within-year occurrence of reported HeV cases, although there were no overall differences in survival in HeV case years and non-case years. The model examines the effect of temperature in isolation; actual virus survivability will reflect the effect of additional environmental factors.

Adaptations of a competitive enzyme-linked immunosorbent assays for the detection of antibodies to influenza a virus in horse sera for use in wild aquatic birds.

We applied a competitive enzyme-linked immunosorbent assay for the detection of antibodies for influenza A in equine sera to their detection in sera from wild aquatic birds. Suboptimal results were obtained for the optical density (OD) of the monoclonal antibody (MAb) control and reproducibility between duplicate analyses in the initial assessment. It was therefore necessary to modify the assay to deliver increased reliability and reproducibility while maintaining adequate sensitivity. We optimized reagent concentrations to obtain optimal OD values (close to 2) for the monoclonal antibody control and used 2, 2'-Azino-bis: 3-Benzthiazoline-6-Sulphonic Acid as an alternative chromogen to potentially reduce variability in duplicate analyses. The original assay was compared with the optimized versions, with and without post coating, for the detection of avian influenza viral antibodies in 240 sera obtained from wild plumed whistling ducks. A separate analytical sensitivity study on diluted positive field sera of plumed whistling ducks and a test of antigen stability after post coating were also performed. Some quantitative differences were detected between the original and modified assays. The original assay recorded higher percentage inhibition results which were potentially indicative of increased sensitivity. However, when reagent concentrations were increased in the original assay to the same levels as used in the modified versions, there were no quantitative differences for practical purposes. The original assay produced a median (OD) value of 0.81 for the (MAb) controls that is at the limit of acceptability. By contrast, the modified assays always produced acceptable optical density values for MAb controls. Our overall results indicated the modified assays were potentially more reliable (OD values close to 2), and of adequate sensitivity compared to the original assay in the detection of avian influenza viral antibodies in wild bird sera. Although further optimization of antigen and MAb concentrations should also be considered to increase the sensitivity of a modified assay, while maintaining acceptable optical density values for the MAb control. Post coating had a minimal quantitative effect on the results and stabilized the plates for 214 days. We therefore recommend the incorporation of post coating.

Diagnosis of equine influenza virus infections in quarantine stations in Australia, 2007.

In August 2007, several horses showed pyrexia and respiratory signs while in post-arrival quarantine in Australia. Subsequent investigations diagnosed equine influenza by serology and PCR in two quarantine stations. A common origin in a shipment of horses from Japan was indicated.

The first five days: field and laboratory investigations during the early stages of the equine influenza outbreak in Australia, 2007.

Until August 2007, Australia was one of only three countries internationally recognised to be free of equine influenza (EI). This report documents the diagnosis of the first cases of EI in Australian horses and summarises the investigations that took place over the next 5 days. During that time, a multifocal outbreak was identified across eastern New South Wales and south-eastern Queensland. The use of an influenza type A pan-reactive real-time reverse transcription polymerase chain reaction allowed rapid confirmation of suspect cases of EI.

Isolation and characterisation of an H3N8 equine influenza virus in Australia, 2007.

Before 2007, equine influenza had never been diagnosed in Australia. On 22 August 2007, infection was confirmed in horses at Eastern Creek Animal Quarantine Station near Sydney. The virus subsequently isolated (A/equine/Sydney/2888-8/2007) was confirmed by sequence analysis of the haemagglutinin (HA) gene as an H3 virus of the variant American Florida lineage that is now referred to as Clade 1. The HA sequence of the virus was identical to that of a virus isolated from a contemporaneous outbreak in Japan and showed high homology to viruses circulating in North America.

Rapid detection of highly pathogenic avian influenza H5N1 virus by TaqMan reverse transcriptase-polymerase chain reaction.

Highly pathogenic avian influenza (AI) H5N1 viruses have been spreading from Asia since late 2003. Early detection and classification are paramount for control of the disease because these viruses are lethal to birds and have caused fatalities in humans. Here, we described TaqMan reverse transcriptase-polymerase chain reaction assays for rapid detection of all AI viruses (influenza type A) and for identification of H5N1 of the Eurasian lineage. The assays were sensitive and quantitative over a 10(5)-10(6) linear range, detected all of the tested AI viruses, and enabled differentiation between H5 and H7 subtypes. These tests allow definitive confirmation of an AI virus as H5 within hours, which is crucial for rapid implementation of control measures in the event of an outbreak.

Susceptibility of highly pathogenic A(H5N1) avian influenza viruses to the neuraminidase inhibitors and adamantanes.

Since 2003, highly pathogenic A(H5N1) influenza viruses have been the cause of large-scale death in poultry and the subsequent infection and death of over 140 humans. A group of 55 influenza A(H5N1) viruses isolated from various regions of South East Asia between 2004 and 2006 were tested for their susceptibility to the anti-influenza drugs the neuraminidase inhibitors and adamantanes. The majority of strains were found to be fully sensitive to the neuraminidase inhibitors oseltamivir carboxylate, zanamivir and peramivir; however two strains demonstrated increased IC50 values. Sequence analysis of these strains revealed mutations in the normally highly conserved residues 116 and 117 of the N1 neuraminidase. Sequence analysis of the M2 gene showed that all of the A(H5N1) viruses from Vietnam, Malaysia and Cambodia contained mutations (L26I and S31N) associated with resistance to the adamantane drugs (rimantadine and amantadine), while strains from Indonesia were found to be a mix of both adamantane resistant (S31N) and sensitive viruses. None of the A(H5N1) viruses from Myanmar contained mutations known to confer adamantane resistance. These results support the use of neuraminidase inhibitors as the most appropriate class of antiviral drug to prevent or treat human A(H5N1) virus infections.

Prion-removal capacity of chromatographic and ethanol precipitation steps used in the production of albumin and immunoglobulins.

BACKGROUND AND OBJECTIVES: Although there is no epidemiological evidence to suggest that classical Creutzfeldt-Jakob disease (CJD) is transmitted through blood or blood products, the variant form (vCJD) has been implicated in transmission via packed red blood cells. The potential threat of the infectious agent contaminating plasma pools has led to manufacturing processes being examined for capacity to remove prions. The objective of these studies was to examine the prion-removal potential of the chromatographic purification and ethanol precipitation steps used to fractionate immunoglobulins and albumin from human plasma. MATERIALS AND METHODS: Western blot assay was used to examine the partitioning of proteinase K-resistant scrapie prion protein (PrPsc) over DEAE Sepharose, CM Sepharose and Macro-Prep High Q chromatographic columns, utilizing microsomal scrapie 263K spiked into each scaled down feedstream and assayed after each chromatographic step. In further studies, bioassay in C57 black mice was used and spikes of 10 000 g clarified brain homogenate of scrapie ME7 were added to feedstreams before sequences of scaled down chromatographic or Cohn fractionation process steps. RESULTS: The microsomal spiking study with Western blot detection demonstrated substantial partitioning of PrPsc away from the target proteins in all ion exchange chromatographic steps examined. The log10 reduction factors (LRF) across DEAE Sepharose and CM Sepharose columns for albumin were > or = 4.0 and > or = 3.0 respectively. The reductions across DEAE Sepharose and Macro-Prep High Q for intravenous immunoglobulin were 3.3 and > or = 4.1 respectively. Bioassay demonstrated LRFs of >or = 5.6 across the combination of DEAE Sepharose and CM Sepharose columns in the albumin process and > or = 5.4 across the combination of DEAE Sepharose and Macro-Prep High Q columns in the intravenous immunoglobulin process. Bioassay studies also demonstrated a LRF of > or = 5.6 for immunoglobulin produced by Cohn fractionation. CONCLUSIONS: Using rodent-adapted scrapie as a model, the studies indicated that ion exchange chromatography, as well as Cohn immunoglobulin fractionation have the potential to effectively reduce the load of TSE agents should they be present in plasma pools.

Isolation of avian influenza viruses from two different transhemispheric migratory shorebird species in Australia.

Shorebirds on their southerly migration from Siberia to Australia, may pass through Asian regions currently experiencing outbreaks of highly pathogenic H5N1 influenza. To test for the presence of avian influenza viruses in migratory shorebirds arriving in Australia during spring 2004, 173 cloacal swabs were collected from six species. Ten swabs were positive for influenza A, with H4N8 viruses detected in five red-necked stints and H11N9 viruses detected in five sharp-tailed sandpipers. No H5N1 viruses were detected. All isolated viruses were non-pathogenic in domestic chickens. These results further demonstrate the potential for migratory shorebirds to carry and potentially spread influenza viruses.

Virus strains from a flock exhibiting unusually high mortality due to infectious bursal disease.

OBJECTIVE: To characterise infectious bursal disease viruses (IBDVs) isolated from commercial broiler flocks exhibiting unusually high mortality due to infectious bursal disease (IBD). DESIGN: An IBD outbreak occurred in mid 1999 on two broilers farms (A and B) in northern New South Wales amongst chickens 28 to 38 days of age, with a sharp rise in mortality of 2.5%. Initial histopathological diagnosis indicated acute IBD. Since acute IBD caused by classical pathogenic and very virulent (vv) IBDVs is exotic to Australia, samples from both farms A and B were obtained and used for virus characterisation. METHOD: Tissue homogenates were made from six bursae collected from farm B. One histological sample from farm A was also used. Nucleotide sequencing of the hypervariable region (HVR) within the VP2 gene of IBDVs was determined and the deduced amino acid sequences compared with previously characterised Australian and overseas IBDVs. The phylogenetic relationship between IBDVs from farm B and IBDVs from Australia and overseas was then determined. Pathogenicity of one isolate, N2/99 from farm B, was compared with 3 other local IBDVs, as well as with three pathogenic overseas strains in 3-week-old specific pathogen-free (SPF) chickens. RESULTS: Initial histopathological characterisation of a sample of bursa from a bird on farm A showed widespread acute lymphoid necrosis, follicular haemorrhage and stromal oedema, indicative of acute IBD. Subsequent analysis using reverse transcriptase polymerase chain reaction (RT-PCR), followed by nucleotide sequencing of the same bursal sample, as well as 6 samples from nearby farm B, showed that the IBDVs involved were similar in sequence to Australian vaccine strains and not to classical pathogenic or vvIBDVs. One isolate, N2/99 from farm B, was only marginally more pathogenic than other local IBDVs. It induced mild clinical signs in 30% of chicks and no mortality. In comparison, vvIBDV CS89 and classical pathogenic 52/70 strains induced severe clinical signs in 100% and 80% of chickens, respectively with mortalities of 27% and 12%, respectively. CONCLUSIONS: The results illustrated the value of nucleotide sequencing as a method for discrimination of local and exotic types of IBDV.

An outbreak of highly pathogenic avian influenza in Australia in 1997 caused by an H7N4 virus.

In November of 1997 an outbreak of highly pathogenic avian influenza occurred near the town of Tamworth, in northern New South Wales, Australia. The viruses isolated from chickens on two commercial chicken farms were identified as H7N4 viruses, with hemagglutinin cleavage site amino acid sequences of RKRKRG and intravenous pathogenicity indices of 2.52 and 2.90, respectively. A virus with an identical nucleotide sequence, but with an intravenous pathogenicity index of 1.30, was also isolated from cloacal swabs collected from asymptomatic emus kept on a third property.

Rapid diagnosis of highly pathogenic avian influenza using pancreatic impression smears.

The 1985 outbreak of high-pathogenicity avian influenza (HPAI) in Victoria, Australia, took 5 days to confirm by standard laboratory tests, during which time infected chickens continued excreting virus, thus creating the opportunity for transmission to other farms. An immunofluorescence test for the detection of viral antigen in tissue impression smears was evaluated as a rapid diagnostic test for HPAI virus infections of poultry. Several test configurations were compared for background reactions and strength of fluorescence, with the optimum combination found to be an influenza A group-specific monoclonal antibody, detected by an anti-mouse fluorescein isothiocyanate conjugate. Immunohistochemical examination of tissues from chickens experimentally infected with low-pathogenicity and HPAI viruses identified the pancreas as the organ most consistently containing high concentrations of HPAI viral antigen. This test has since been used in Australia in the rapid laboratory confirmation of three avian influenza outbreaks and in showing that numerous other suspect cases were not caused by avian influenza.

First identification of a ranavirus from green pythons (Chondropython viridis).

Ten juvenile green pythons (Chondropython viridis) died or were euthanized shortly after having been illegally imported into Australia from Indonesia in 1998. Histologic examination of two of the three snakes that died revealed moderately severe chronic ulceration of the nasal mucosa and focal or periacinar degeneration and necrosis of the liver. In addition there was severe necrotizing inflammation of the pharyngeal submucosa accompanied by numerous macrophages, heterophils, and edema. An iridovirus was isolated in culture from several tissues and characterized by immunohistochemistry, electron microscopy, enzyme-linked immunosorbent Assay, polyacrylamide gel electrophoresis, polymerase chain reaction and sequence analysis, restriction endonuclease digestion, and DNA hybridization. This is the first report of a systemic ranavirus infection in any species of snake and is a new member of the genus, Ranavirus.

A guinea-pig model of Hendra virus encephalitis.

Subcutaneous inoculation, but not intradermal (footpad) or intranasal inoculation, with high doses of Hendra virus (HeV) consistently produced disease in guinea-pigs. Of 15 subcutaneously inoculated animals, 14 developed vascular disease with positive HeV immunohistochemical labelling in a range of tissues. A new observation was the presence of lesions, including syncytial cells, with immunolabelling in the transitional epithelium of the bladder. Virus isolation from the urine rather than from nasal, oral, rectal or conjunctival swabs, the other external sites, was consistent with previous epidemiological work in horses, indicating a limited possibility of transmission. The dose used (30 000 to 50 000 TCID(50)), which was higher than in previous studies, produced microscopical lesions of encephalitis in eight of the 15 subcutaneously inoculated guinea-pigs, with positive immunolabelling in blood vessels and neurons, especially in the medulla, cerebellum and thalamus. The virus was recovered from six of the encephalitic brains. Severe vascular degeneration in the centres of encephalitic lesions in six of the eight encephalitic guinea-pigs and positive immunolabelling in the choroid plexus of a further animal indicated that the virus entered the brain following virus-induced vascular injury and choroid plexus invasion. Guinea-pigs would appear to be suitable for the study of HeV encephalitis.

Virulent Newcastle disease in Australia: molecular epidemiological analysis of viruses isolated prior to and during the outbreaks of 1998-2000.

Gene sequence analysis of fusion (F) gene cleavage motifs and haemagglutinin-neuraminidase (HN) carboxyl-terminal extension sequences was used to analyse Newcastle disease viruses (NDV) associated with virulent outbreaks of the disease which occurred in New South Wales, Australia in 1998-2000. PCR fragments were amplified directly from diseased tissue or allantoic fluids and sequence analyses used for phylogenetic comparisons between these viruses and Australian reference NDV. F and HN gene sequence comparison showed a strong relationship to sequences derived from endemic Australian NDV rather than those of overseas viruses or wild bird isolates. Prior to notification of the 1998 outbreak, an NDV was isolated from chickens suffering respiratory disease that appeared to be the progenitor virus from which the virulent virus originated. In turn, these viruses are closely related to two previously isolated 'ancestor' viruses that have the same unique HN extension sequence.

Vaccinating chickens against avian influenza with fowlpox recombinants expressing the H7 haemagglutinin.

OBJECTIVE: To evaluate the vaccine efficacy of a fowlpox virus recombinant expressing the H7 haemagglutinin of avian influenza virus in poultry. PROCEDURE: Specific-pathogen-free poultry were vaccinated with fowlpox recombinants expressing H7 or H1 haemagglutinins of influenza virus. Chickens were vaccinated at 2 or 7 days of age and challenged with virulent Australian avian influenza virus at 10 and 21 days later, respectively. Morbidity and mortality, body weight change and the development of immune responses to influenza haemagglutinin and nucleoprotein were recorded. RESULTS: Vaccination of poultry with fowlpox H7 avian influenza virus recombinants induced protective immune responses. All chickens vaccinated at 7 days of age and challenged 21 days later were protected from death. Few clinical signs of infection developed. In contrast, unvaccinated or chickens vaccinated with a non-recombinant fowlpox or a fowlpox expressing the H1 haemagglutinin of human influenza were highly susceptible to avian influenza. All those chickens died within 72 h of challenge. In younger chickens, vaccinated at 2 days of age and challenged 10 days later the protection was lower with 80% of chickens protected from death. Chickens surviving vaccination and challenge had high antibody responses to haemagglutinin and primary antibody responses to nucleoprotein suggesting that although vaccination protected substantially against disease it failed to completely prevent replication of the challenge avian influenza virus. CONCLUSION: Vaccination of chickens with fowlpox virus expressing the avian influenza H7 haemagglutinin provided good protection against experimental challenge with virulent avian influenza of H7 type. Although eradication will remain the method of first choice for control of avian influenza, in the circumstances of a continuing and widespread outbreak the availability of vaccines based upon fowlpox recombinants provides an additional method for disease control.