Detection and stability of Japanese encephalitis virus RNA and virus viability in dead infected mosquitoes under different storage conditions

A semi-nested polymerase chain reaction (PCR) was evaluated for detection of Japanese encephalitis (JE) virus in infected mosquitoes stored under simulated northern Australian summer conditions. The effect of silica gel, thymol, and a combination of the two on RNA stability and virus viability in dead mosquitoes were also examined. While JE virus RNA was relatively stable in mosquitoes held for up to 14 days after death, viable virus was not detected after day 1. Thymol vapor inhibited fungal contamination. Detection of single mosquitoes infected with JE virus in large pools of mosquitoes was also investigated. Single laboratory-infected mosquitoes were detected in pools of less than or equal to200 mosquitoes and in pools diluted to 0.2/100 and 0.1/100 mosquitoes, using the semi-nested PCR. However, the ability to detect live virus decreased as pool size increased. The semi-nested PCR proved more expensive than virus isolation for pools of 100 mosquitoes. However, the semi-nested PCR was faster and more economical using larger pools. Results indicate that surveillance of JE virus in mosquitoes using the semi-nested PCR is an alternative to monitoring seroconversions in sentinel pigs.

Air target detection via bistatic radar based on LEOS communication signals

The paper discusses the bistatic radar parameters for the case when the transmitter is a satellite emitting communication signals. The model utilises signals from an Iridium-like low earth orbiting satellite system. The maximum detection range, when thermal noise-limited, is discussed at the theoretical level and these results are compared with experimentation. Satellite-radar signal levels and the power of ground reflections are evaluated.

Noninvasive measurement of cerebral bioimpedance for detection of cerebral edema in the neonatal piglet

The association of sustained cerebral edema with poor neurological outcome following hypoxia-ischaemia in the neonate suggests that measurement of cerebral edema may allow early prediction of outcome in these infants. Direct measurements of cerebral impedance have been widely used in animal studies to monitor cerebral edema, but such invasive measurements are not possible in the human neonate. This study investigated the ability of noninvasive cerebral impedance measurements to detect cerebral edema following hypoxia-ischaemia. One-day-old piglets were anaesthetized, intubated and ventilated. Hypoxia was induced by reducing the inspired oxygen concentration to 4-6% O-2. Noninvasive cerebral bioimpedance was measured using gel electrodes attached to the scalp. Cerebral bioimpedance was also measured directly by insertion of two silver-silver chloride electrodes subdurally. Noninvasive and invasive measurements were made before, during and after hypoxia. Whole body impedance was measured to assess overall fluid movements. Intracranial pressure was measured continuously via a catheter inserted subdurally, as an index of cerebral edema. There was good agreement between noninvasive and invasive measurements of cerebral impedance although externally obtained responses were attenuated. Noninvasive measurements were also well correlated with intracranial pressure. Whole body impedance changes did not account for increases in noninvasively measured cerebral impedance. Results suggest that noninvasive cerebral impedance measurements do reflect intracranial events, and are able to detect cerebral edema following hypoxia-ischaemia in the neonate. (C) 2002 Elsevier Science B.V. All rights reserved.

Specificity of PCR and in situ hybridization assays designed for detection of Marteilia sydneyi and M-refringens

Primers and DNA probes designed for use in the specific detection of the paramyxean parasites Marteilia sydneyi and Marteilia refringens were tested for their potential to cross-react with closely related species in Polymerase Chain Reaction (PCR) and in situ hybridization. PCR primers and a DNA probe designed within the ITS1 rRNA of M. sydneyi were specific for M. sydneyi when compared with related species of Marteilia and Marteilioides. PCR primers designed within the 18S rRNA of M. refringens were specific in the detection of this species in PCR while a DNA probe (named Smart 2) designed on the same gene cross-reacted with M. sydneyi in tissue sections of Saccostrea glomerata as well as Marteilioides sp. infecting Striostrea mytiloides. Though not species specific, the Smart 2 probe provided a stronger signal in detection of all stages of M. sydneyi than the ITS1 probe. The ITS probe is proposed for use as a confirmatory diagnostic too] for M. sydneyi.

Detection of the initial infective stages of the protozoan parasite Marteilia sydneyi in Saccostrea glomerata and their development through to sporogenesis

DNA probes were used in in situ hybridisation on histological sections of oysters exposed for defined intervals to Marteilia sydneyi infection to reveal the early development of the parasite in the oyster host, Saccostrea glomerata. The initial infective stages enter through the palps and gills whereupon extrasporogonic proliferation results in the liberation of cells into surrounding connective tissue and haemolymph spaces. Following systemic dissemination, the parasite infiltrates the digestive gland and becomes established as a nurse cell beneath the epithelial cells ill a digestive tubule. Here, cell-within-cell proliferation results in the eventual liberation of daughter cells from the nurse cell into spaces between adjacent epithelial cells. None of these stages had previously been described. Proliferation is associated with host responses, including haemocytic infiltration of the connective tissue and diapedesis across tubule epithelia. The responses cease as sporogenesis begins. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.

The development of a positive non-infectious control for the detection of Perkinsus using the Ray test

To establish a noncontagious control for the Ray thioglycollate test for the detection of Perkinsus in mollusks we evaluated nonviable stages of P. olseni for enlargement of hypnospores and blue/black iodine stain. Trophozoites made nonviable with formalin, irradiation or colchicine failed to swell in thioglycollate. They remained small and did not differentially stain in iodine. Trophozoites that had already developed into hypnospores in thioglycollate were rendered inactive by freezing, ethanol or formalin immersion. They retained their iodinophilic properties and thus could provide a partial control for the Ray Test.

Detection of swallowing sounds: Methodology revisited

Cervical auscultation is in the process of gaining clinical credibility. In order for it to be accepted by the clinical community, the procedure and equipment used must first be standardized. Takahashi et al. [Dysphagia 9:54-62, 1994] attempted to provide benchmark methodology for administering cervical auscultation. They provided information about the acoustic detector unit best suited to picking up swallowing sounds and the best cervical site to place it. The current investigation provides contrasting results to Takahashi et al. with respect to the best type of acoustic detector unit to use for detecting swallowing sounds. Our study advocates an electret microphone as opposed to an accelerometer for recording swallowing sounds. However, we agree on the optimal placement site. We conclude that cervical auscultation is within reach of the average dysphagia clinic.

Patterns of resistance to angular leaf spot, anthracnose and common bacterial blight in common bean germplasm

Diseases and insect pests are major causes of low yields of common bean (Phaseolus vulgaris L.) in Latin America and Africa. Anthracnose, angular leaf spot and common bacterial blight are widespread foliar diseases of common bean that also infect pods and seeds. One thousand and eighty-two accessions from a common bean core collection from the primary centres of origin were investigated for reaction to these three diseases. Angular leaf spot and common bacterial blight were evaluated in the field at Santander de Quilichao, Colombia, and anthracnose was evaluated in a screenhouse in Popayan, Colombia. By using the 15-group level from a hierarchical clustering procedure, it was found that 7 groups were formed with mainly Andean common bean accessions (Andean gene pool), 7 groups with mainly Middle American accessions (Middle American gene pool), while 1 group contained mixed accessions. Consistent with the theory of co-evolution, it was generally observed that accessions from the Andean gene pool were resistant to Middle American pathogen isolates causing anthracnoxe, while the Middle American accessions were resistant to pathogen isolates from the Andes. Different combinations of resistance patterns were found, and breeders can use this information to select a specific group of accessions on the basis of their need.

Sensitivity and specificity of pooled faecal culture and serology as flock-screening tests for detection of ovine paratuberculosis in Australia

The flock-level sensitivity of pooled faecal culture and serological testing using AGID for the detection of ovine Johne's disease-infected flocks were estimated using non-gold-standard methods. The two tests were compared in an extensive field trial in 296 flocks in New South Wales during 1998. In each flock, a sample of sheep was selected and tested for ovine Johne's disease using both the AGID and pooled faecal culture. The flock-specificity of pooled faecal culture also was estimated from results of surveillance and market-assurance testing in New South Wales. The overall flock-sensitivity of pooled faecal culture was 92% (95% CI: 82.4 and 97.4%) compared to 61% (50.5 and 70.9%) for serology (assuming that both tests were 100% specific). In low-prevalence flocks (estimated prevalence

A rapid PCR protocol for marker assisted detection of heterozygotes in segregating generations involving 1BL/1RS translocation and normal wheat lines

A rapid and reliable polymerase chain reaction (PCR)-based protocol was developed for detecting zygosity of the 1BL/1RS translocation in hexaploid wheat. The protocol involved a multiplex PCR with 2 pairs of oligonucleotide primers, rye-specific Ris-1 primers, and consensus 5S intergenic spacer (IGS) primers, and digestion of the PCR products with the restriction enzyme, MseI. A small piece of alkali-treated intact leaf tissue is used as a template for the PCR, thereby eliminating the necessity for DNA extraction. The test is simple, highly sensitive, and rapid compared with the other detection systems of 1BS1RS heterozygotes in hexaploid wheat. PCR results were confirmed with AFLP analyses. Diagnostic tests for 1BL/1RS translocation based on Sec-1-specific ELISA, screening for chromosome arm 1RS controlled rust resistance locus Yr9, and the PCR test differed in their ability to detect heterozygotes. The PCR test and rust test detected more heterozygotes than the ELISA test. The PCR test is being used to facilitate S1 family recurrent selection in the Germplasm Enhancement Program of the Australian Northern Wheat Improvement Program. A combination of the PCR zygosity test with other markers currently being implemented in the breeding program makes this test economical for 1BL/1RS characterisation of S1 families.

Distributional profiles of homologous open reading frames among bacterial phyla: implications for vertical and lateral transmission

If open reading frames (ORFs) have been transmitted primarily by vertical descent, the distributional profile of orthologues of each ORF should be congruent with the organismal tree or a subtree thereof. Distributional patterns not reconciled parsimoniously with tree-like descent and loss are prima facie evidence of lateral gene transfer. Herein, a rigorous criterion for recognizing ORF distributions is described and implemented; it does not require the inference of phylogenetic trees, nor does it assume any specific tree. Because lineage-specific differences in rates of sequence change can also generate unexpected distributional patterns, rate artefacts, were controlled for by requiring pairwise matches between ORFs to exceed a rigorous inclusion threshold, but absence of a match was assessed against a more-permissive exclusion threshold. Applying this dual-threshold criterion to cross-domain and cross-phylum distributional patterns for ORFs in 23 bacterial genomes, a relative abundance of ORFs was observed that find a match in exactly seven other bacterial phyla; 94-99% of these ORFs also find matches among the Archaea and/or Eukarya. In the larger (and some smaller) bacterial genomes, ORFs that find matches in exactly one other bacterial phylum are also relatively abundant, but fewer of these have non-bacterial homologues; most of their matches within the Bacteria are to the Proteobacteria and/or Firmicutes, which cannot be sister lineages to all bacteria. ORFs that are neither distributed universally among the Bacteria, nor necessarily shared with topologically adjacent lineages, are preferentially enriched in large bacterial genomes.