Potential Animal and Environmental Sources of Q Fever Infection for Humans in Queensland

Q fever is a vaccine-preventable disease; despite this, high annual notification numbers are still recorded in Australia. We have previously shown seroprevalence in Queensland metropolitan regions is approaching that of rural areas. This study investigated the presence of nucleic acid from Coxiella burnetii, the agent responsible for Q fever, in a number of animal and environmental samples collected throughout Queensland, to identify potential sources of human infection. Samples were collected from 129 geographical locations and included urine, faeces and whole blood from 22 different animal species; 45 ticks were removed from two species, canines and possums; 151 soil samples; 72 atmospheric dust samples collected from two locations and 50 dust swabs collected from domestic vacuum cleaners. PCR testing was performed targeting the IS1111 and COM1 genes for the specific detection of C.burnetii DNA. There were 85 detections from 1318 animal samples, giving a detection rate for each sample type ranging from 2.1 to 6.8%. Equine samples produced a detection rate of 11.9%, whilst feline and canine samples showed detection rates of 7.8% and 5.2%, respectively. Native animals had varying detection rates: pooled urines from flying foxes had 7.8%, whilst koalas had 5.1%, and 6.7% of ticks screened were positive. The soil and dust samples showed the presence of C.burnetii DNA ranging from 2.0 to 6.9%, respectively. These data show that specimens from a variety of animal species and the general environment provide a number of potential sources for C.burnetii infections of humans living in Queensland. These previously unrecognized sources may account for the high seroprevalence rates seen in putative low-risk communities, including Q fever patients with no direct animal contact and those subjects living in a low-risk urban environment.

Applications of a sugar-based surveillance system to track arboviruses in wild mosquito populations

Effective arbovirus surveillance is essential to ensure the implementation of control strategies, such as mosquito suppression, vaccination, or dissemination of public warnings. Traditional strategies employed for arbovirus surveillance, such as detection of virus or virus-specific antibodies in sentinel animals, or detection of virus in hematophagous arthropods, have limitations as an early-warning system. A system was recently developed that involves collecting mosquitoes in CO2-baited traps, where the insects expectorate virus on sugar-baited nucleic acid preservation cards. The cards are then submitted for virus detection using molecular assays. We report the application of this system for detecting flaviviruses and alphaviruses in wild mosquito populations in northern Australia. This study was the first to employ nonpowered passive box traps (PBTs) that were designed to house cards baited with honey as the sugar source. Overall, 20/144 (13.9%) of PBTs from different weeks contained at least one virus-positive card. West Nile virus Kunjin subtype (WNVKUN), Ross River virus (RRV), and Barmah Forest virus (BFV) were detected, being identified in 13/20, 5/20, and 2/20 of positive PBTs, respectively. Importantly, sentinel chickens deployed to detect flavivirus activity did not seroconvert at two Northern Territory sites where four PBTs yielded WNVKUN. Sufficient WNVKUN and RRV RNA was expectorated onto some of the honey-soaked cards to provide a template for gene sequencing, enhancing the utility of the sugar-bait surveillance system for investigating the ecology, emergence, and movement of arboviruses. © 2014, Mary Ann Liebert, Inc.

Isolation of multiple novel paramyxoviruses from pteropid bat urine

Bats have been found to harbor a number of new emerging viruses with zoonotic potential and there has been a great deal of interest in identifying novel bat pathogens to determine risk to human and animal health. Many groups have identified novel viruses in bats by detection of viral nucleic acid, however virus isolation is still a challenge and there are few reports of viral isolates from bats. In recent years, our group has developed optimized procedures for virus isolation from bat urine, including the use of primary bat cells. In previous reports we have described the isolation of Hendra virus, Menangle virus and Cedar virus, in Queensland, Australia. Here, we report the isolation of four additional novel bat paramyxoviruses from urine collected from beneath pteropid bat (flying fox) colonies in Queensland and New South Wales during 2009-2011.

New yellow head virus genotype (YHV7) in giant tiger shrimp Penaeus monodon indigenous to northern Australia

ABSTRACT: In 2012, giant tiger shrimp Penaeus monodon originally sourced from Joseph Bonaparte Gulf in northern Australia were examined in an attempt to identify the cause of elevated mortalities among broodstock at a Queensland hatchery. Nucleic acid extracted from ethanol-fixed gills of 3 individual shrimp tested positive using the OIE YHV Protocol 2 RT-PCR designed to differentiate yellow head virus (YHV1) from gill-associated virus (GAV, synonymous with YHV2) and the OIE YHV Protocol 3 RT-nested PCR designed for consensus detection of YHV genotypes. Sequence analysis of the 794 bp (Protocol 2) and 359 bp (Protocol 3) amplicons from 2 distinct regions of ORF1b showed that the yellow-head-complex virus detected was novel when compared with Genotypes 1 to 6. Nucleotide identity on the Protocol 2 and Protocol 3 ORF1b sequences was highest with the highly pathogenic YHV1 genotype (81 and 87%, respectively) that emerged in P. monodon in Thailand and lower with GAV (78 and 82%, respectively) that is enzootic to P. monodon inhabiting eastern Australia. Comparison of a longer (725 bp) ORF1b sequence, spanning the Protocol 3 region and amplified using a modified YH30/31 RT-nPCR, provided further phylogenetic evidence for the virus being distinct from the 6 described YHV genotypes. The virus represents a unique seventh YHV genotype (YHV7). Despite the mortalities observed, the role of YHV7 remains unknown.

Molecular evidence of Ebola Reston virus infection in Philippine bats

Background: In 2008-09, evidence of Reston ebolavirus (RESTV) infection was found in domestic pigs and pig workers in the Philippines. With species of bats having been shown to be the cryptic reservoir of filoviruses elsewhere, the Philippine government, in conjunction with the Food and Agriculture Organization of the United Nations, assembled a multi-disciplinary and multi-institutional team to investigate Philippine bats as the possible reservoir of RESTV. Methods: The team undertook surveillance of bat populations at multiple locations during 2010 using both serology and molecular assays. Results: A total of 464 bats from 21 species were sampled. We found both molecular and serologic evidence of RESTV infection in multiple bat species. RNA was detected with quantitative PCR (qPCR) in oropharyngeal swabs taken from Miniopterus schreibersii, with three samples yielding a product on conventional hemi-nested PCR whose sequences differed from a Philippine pig isolate by a single nucleotide. Uncorroborated qPCR detections may indicate RESTV nucleic acid in several additional bat species (M. australis, C. brachyotis and Ch. plicata). We also detected anti-RESTV antibodies in three bats (Acerodon jubatus) using both Western blot and ELISA. Conclusions: The findings suggest that ebolavirus infection is taxonomically widespread in Philippine bats, but the evident low prevalence and low viral load warrants expanded surveillance to elaborate the findings, and more broadly, to determine the taxonomic and geographic occurrence of ebolaviruses in bats in the region. © 2015 Jayme et al.

A comparison of a blocking ELISA and a haemagglutination inhibition assay for the detection of antibodies to Avibacterium (Haemophilus) paragallinarum in sera from artificially infected chickens

The ability of blocking ELISAs and haemagglutination-inhibition (HI) tests to detect antibodies in sera from chickens challenged with either Avibacterium (Haemophilus) paragallinarum isolate Hp8 (serovar A) or H668 (serovar C) was compared. Serum samples were examined weekly over the 9 weeks following infection. The results showed that the positive rate of serovar A specific antibody in the B-ELISA remained at 100% from the second week to the ninth week. In chickens given the serovar C challenge, the highest positive rate of serovar C specific antibody in the B-ELISA appeared at the seventh week (60% positive) and was then followed by a rapid decrease. The B-ELISA gave significantly more positives at weeks 2, 3, 7, 8 and 9 post-infection for serovar A and at week 7 post-infection for serovar C. In qualitative terms, for both serovar A and serovar C infections, the HI tests gave a lower percentage of positive sera at all time points except at 9 weeks post-infection with serovar C. The highest positive rate for serovar A HI antibodies was 70% of sera at the fourth and fifth weeks post-infection. The highest rate of serovar C HI antibodies was 20% at the fifth and sixth weeks post-infection. The results have provided further evidence of the suitability of the serovar A and C B-ELISAs for the diagnosis of infectious coryza.

Availability of soil potassium and diagnostic soil tests

Thirty-seven surface (0-0.10 or 0-0.20 m) soils covering a wide range of soil types (16 Vertosols, 6 Ferrosols, 6 Dermosols, 4 Hydrosols, 2 Kandosols, 1 Sodosol, 1 Rudosol, and 1 Chromosol) were exhaustively cropped in 2 glasshouse experiments. The test species were Panicum maximum cv. Green Panic in Experiment A and Avena sativa cv. Barcoo in Experiment B. Successive forage harvests were taken until the plants could no longer grow in most soils because of severe potassium (K) deficiency. Soil samples were taken prior to cropping and after the final harvest in both experiments, and also after the initial harvest in Experiment B. Samples were analysed for solution K, exchangeable K (Exch K), tetraphenyl borate extractable K for extraction periods of 15 min (TBK15) and 60 min (TBK60), and boiling nitric acid extractable K (Nitric K). Inter-correlations between the initial levels of the various soil K parameters indicated that the following pools were in sequential equilibrium: solution K, Exch K, fast release fixed K [estimated as (TBK15-Exch K)], and slow release fixed K [estimated as (TBK60-TBK15)]. Structural K [estimated as (Nitric K-TBK60)] was not correlated with any of the other pools. However, following exhaustive drawdown of soil K by cropping, structural K became correlated with solution K, suggesting dissolution of K minerals when solution K was low. The change in the various K pools following cropping was correlated with K uptake at Harvest 1 ( Experiment B only) and cumulative K uptake ( both experiments). The change in Exch K for 30 soils was linearly related to cumulative K uptake (r = 0.98), although on average, K uptake was 35% higher than the change in Exch K. For the remaining 7 soils, K uptake considerably exceeded the change in Exch K. However, the changes in TBK15 and TBK60 were both highly linearly correlated with K uptake across all soils (r = 0.95 and 0.98, respectively). The slopes of the regression lines were not significantly different from unity, and the y-axis intercepts were very small. These results indicate that the plant is removing K from the TBK pool. Although the change in Exch K did not consistently equate with K uptake across all soils, initial Exch K was highly correlated with K uptake (r = 0.99) if one Vertosol was omitted. Exchangeable K is therefore a satisfactory diagnostic indicator of soil K status for the current crop. However, the change in Exch K following K uptake is soil-dependent, and many soils with large amounts of TBK relative to Exch K were able to buffer changes in Exch K. These soils tended to be Vertosols occurring on floodplains. In contrast, 5 soils (a Dermosol, a Rudosol, a Kandosol, and 2 Hydrosols) with large amounts of TBK did not buffer decreases in Exch K caused by K uptake, indicating that the TBK pool in these soils was unavailable to plants under the conditions of these experiments. It is likely that K fertiliser recommendations will need to take account of whether the soil has TBK reserves, and the availability of these reserves, when deciding rates required to raise exchangeable K status to adequate levels.

Detection of Polymyxa graminis in a barley crop in Australia.

Polymyxa graminis was detected in the roots of barley plants from a field near Wondai, Queensland, in 2009. P. graminis was identified by characteristic sporosori in roots stained with trypan blue. The presence of P. graminis f. sp. tepida (which is hosted by wheat and oats as well as barley) in the roots was confirmed by specific PCR tests based on nuclear ribosomal DNA. P. graminis is the vector of several damaging soil-borne virus diseases of cereals in the genera Furovirus, Bymovirus and Pecluvirus. No virus particles were detected in sap extracts from leaves of stunted barley plants with leaf chlorosis and increased tillering. Further work is required to determine the distribution of P. graminis in Australian grain crops and the potential for establishment and spread of the exotic soil-borne viruses that it vectors.

Identification of a new Polerovirus (family Luteoviridae) associated with cotton bunchy top disease in Australia

Cotton bunchy top (CBT) disease has caused significant yield losses in Australia and is now managed by control of its vector, the cotton aphid (Aphis gossypii). Its mode of transmission and similarities in symptoms to cotton Blue Disease suggested it may also be caused by a luteovirus or related virus. Degenerate primers to conserved regions of the genomes of the family Luteoviridae were used to amplify viral cDNAs from CBT-affected cotton leaf tissue that were not present in healthy plants. Partial genome sequence of a new virus (Cotton bunchy top virus, CBTV) was obtained spanning part of the RNA-dependent-RNA-polymerase (RdRP), all of the coat protein and part of the aphid-transmission protein. CBTV sequences could be detected in viruliferous aphids able to transmit CBT, but not aphids from non-symptomatic plants, indicating that it is associated with the disease and may be the causal agent. All CBTV open-reading frames had their closest similarity to viruses of the genus Polerovirus. The partial RdRP had 90 % amino acid identity to the RdRP of Cotton leafroll dwarf virus (CLRDV) that causes cotton blue disease, while other parts of the genome were more similar to other poleroviruses. The sequence similarity and genome organization of CBTV suggest that it should be considered a new member of the genus Polerovirus. This partial genome sequence of CBTV opens up the possibility for developing diagnostic tests for detection of the virus in cotton plants, aphids and weeds as well as alternative strategies for engineering CBT resistance in cotton plants through biotechnology. © 2012 Australasian Plant Pathology Society Inc.

A simple, one-tube assay for the simultaneous detection and diagnosis of ten Australian poultry Eimeria

Coccidiosis is a costly worldwide enteric disease of chickens caused by parasites of the genus Eimeria. At present, there are seven described species that occur globally and a further three undescribed, operational taxonomic units (OTUs X, Y, and Z) that are known to infect chickens from Australia. Species of Eimeria have both overlapping morphology and pathology and frequently occur as mixed-species infections. This makes definitive diagnosis with currently available tests difficult and, to date, there is no test for the detection of the three OTUs. This paper describes the development of a PCR-based assay that is capable of detecting all ten species of Eimeria, including OTUs X, Y, and Z in field samples. The assay is based on a single set of generic primers that amplifies a single diagnostic fragment from the mitochondrial genome of each species. This one-tube assay is simple, low-cost, and has the capacity to be high throughput. It will therefore be of great benefit to the poultry industry for Eimeria detection and control, and the confirmation of identity and purity of vaccine strains.

Measuring free ellagic acid: influence of extraction conditions on recovery by studying solubility and UV-Visible spectra

Recent investigations into plant tissues have indicated that the free form of the natural polyphenolic antioxidant, ellagic acid (EA), is much more plentiful than first envisaged; consequently a re-assessment of solvent systems for the extraction of this water-insoluble form is needed. As EA solubility and its UV-Vis spectrum, commonly used for detection and quantification, are both governed by pH, an understanding of this dependence is vital if accurate EA measurements are to be achieved. After evaluating the pH effects on the solubility and UV-Vis spectra of commercial EA, an extraction protocol was devised that promoted similar pH conditions for both standard solutions and plant tissue extracts. The extraction so devised followed by HPLC with photodiode-array detection (DAD) provided a simple, sensitive and validated methodology that determined free EA in a variety of plant extracts. The use of 100 % methanol or a triethanolamine-based mixture as the standard dissolving solvents were the best choices, while these higher pH-generating solvents were more efficient in extracting EA from the plants tested with the final choice allied to the plants’ natural acidity. Two of the native Australian plants anise myrtle (Syzygium anisatum) and Kakadu plum (Terminalia ferdinandiana) exhibited high concentrations of free EA. Furthermore, the dual approach to measuring EA UV-Vis spectra made possible an assessment of the effect of acidified eluent on EA spectra when the DAD was employed.