Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4).

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB?) labelled with 6-carboxy-fluorescein (FAM?) and VIC® for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.

Completion of the genome sequence of Lettuce necrotic yellows virus, type species of the genus Cytorhabdovirus

We completed the genome sequence of Lettuce necrotic yellows virus (LNYV) by determining the nucleotide sequences of the 4a (putative phosphoprotein), 4b, M (matrix protein), G (glycoprotein) and L (polymerase) genes. The genome consists of 12,807 nucleotides and encodes six genes in the order 3′ leader-N-4a(P)-4b-M-G-L-5′ trailer. Sequences were derived from clones of a cDNA library from LNYV genomic RNA and from fragments amplified using reverse transcription-polymerase chain reaction. The 4a protein has a low isoelectric point characteristic for rhabdovirus phosphoproteins. The 4b protein has significant sequence similarities with the movement proteins of capillo- and trichoviruses and may be involved in cell-to-cell movement. The putative G protein sequence contains a predicted 25 amino acids signal peptide and endopeptidase cleavage site, three predicted glycosylation sites and a putative transmembrane domain. The deduced L protein sequence shows similarities with the L proteins of other plant rhabdoviruses and contains polymerase module motifs characteristic for RNA-dependent RNA polymerases of negative-strand RNA viruses. Phylogenetic analysis of this motif among rhabdoviruses placed LNYV in a group with other sequenced cytorhabdoviruses, most closely related to Strawberry crinkle virus.

Baseline responses of Alphitobius diaperinus (Coleoptera : Tenebrionidae) to cyfluthrin and detection of strong resistance in field populations in eastern Australia

Resistance to cyfluthrin in broiler farm populations of lesser mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), in eastern Australia was suspected to have contributed to recent control failures. In 2000-2001, beetles from 11 broiler farms were tested for resistance by comparing them to an insecticide-susceptible reference population by using topical application. Resistance was detected in almost all beetle populations (up to 22 times the susceptible at the LC50), especially in southeastern Queensland where more cyfluthrin applications had been made. Two from outside southeastern Queensland were found to be susceptible. Dose-mortality data generated from the reference population over a range of cyflutbrin concentrations showed that 0.0007% cyfluthrin at a LC99.9 level could be used as a convenient dose to discriminate between susceptible and resistant populations. Using this discriminating concentration, from 2001 to 2005, the susceptibilities of 18 field populations were determined. Of these, 11 did not exhibit complete mortality at the discriminating concentration (mortality range 2.8-97.7%), and in general, cyfluthrin resistance was directly related to the numbers of cyfluthrin applications. As in the full study, populations outside of southeastern Queensland were found to have lower levels of resistance or were susceptible. One population from an intensively farmed broiler area in southeastern Queensland exhibited low mortality despite having no known exposure to cyfluthrin. Comparisons of LC50 values of three broiler populations and a susceptible population, collected in 2000 and 2001 and recollected in 2004 and 2005 indicated that values from the three broiler populations had increased over this time for all populations. The continued use of cyfluthrin for control of A. diaperinus in eastern Australia is currently under consideration.

Genetic analysis of foot-and-mouth disease virus serotype A of Indian origin and detection of positive selection and recombination in leader protease- and capsid-coding regions

The leader protease (L-pro) and capsid-coding sequences (P1) constitute approximately 3 kb of the foot-and-mouth disease virus (FMDV). We studied the phylogenetic relationship of 46 FMDV serotype A isolates of Indian origin collected during the period 1968-2005 and also eight vaccine strains using the neighbour-joining tree and Bayesian tree methods. The viruses were categorized under three major groups - Asian, Euro-South American and European. The Indian isolates formed a distinct genetic group among the Asian isolates. The Indian isolates were further classified into different genetic subgroups (<5% divergence). Post-1995 isolates were divided into two subgroups while a few isolates which originated in the year 2005 from Andhra Pradesh formed a separate group. These isolates were closely related to the isolates of the 1970s. The FMDV isolates seem to undergo reverse mutation or onvergent evolution wherein sequences identical to the ancestors are present in the isolates in circulation. The eight vaccine strains included in the study were not related to each other and belonged to different genetic groups. Recombination was detected in the L-pro region in one isolate (A IND 20/82) and in the VP1 coding 1D region in another isolate (A RAJ 21/96). Positive selection was identified at aa positions 23 in the L-pro (P<0.05; 0.046*) and at aa 171 in the capsid protein VP1 (P<0.01; 0.003**).

Large cryptic internal sequence repeats in protein structures from Homo sapiens

Amino acid sequences are known to constantly mutate and diverge unless there is a limiting condition that makes such a change deleterious. However, closer examination of the sequence and structure reveals that a few large, cryptic repeats are nevertheless sequentially conserved. This leads to the question of why only certain repeats are conserved at the sequence level. It would be interesting to find out if these sequences maintain their conservation at the three-dimensional structure level. They can play an active role in protein and nucleotide stability, thus not only ensring proper functioning but also potentiating malfunction and disease. Therefore, insights into any aspect of the repeats - be it structure, function or evolution - would prove to be of some importance. This study aims to address the relationship between protein sequence and its three-dimensional structure, by examining if large cryptic sequence repeats have the same structure.

Repeatable Condition-Invariant Visual Odometry for Sequence-Based Place Recognition

This paper describes a vision-only system for place recognition in environments that are tra- versed at different times of day, when chang- ing conditions drastically affect visual appear- ance, and at different speeds, where places aren’t visited at a consistent linear rate. The ma- jor contribution is the removal of wheel-based odometry from the previously presented algo- rithm (SMART), allowing the technique to op- erate on any camera-based device; in our case a mobile phone. While we show that the di- rect application of visual odometry to our night- time datasets does not achieve a level of perfor- mance typically needed, the VO requirements of SMART are orthogonal to typical usage: firstly only the magnitude of the velocity is required, and secondly the calculated velocity signal only needs to be repeatable in any one part of the environment over day and night cycles, but not necessarily globally consistent. Our results show that the smoothing effect of motion constraints is highly beneficial for achieving a locally consis- tent, lighting-independent velocity estimate. We also show that the advantage of our patch-based technique used previously for frame recogni- tion, surprisingly, does not transfer to VO, where SIFT demonstrates equally good performance. Nevertheless, we present the SMART system us- ing only vision, which performs sequence-base place recognition in extreme low-light condi- tions where standard 6-DOF VO fails and that improves place recognition performance over odometry-less benchmarks, approaching that of wheel odometry.

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.

Phylogenetic relationships of graminicolous downy mildews based on cox2 sequence data

Graminicolous downy mildews (GDM) are an understudied, yet economically important, group of plant pathogens, which are one of the major constraints to poaceous crops in the tropics and subtropics. Here we present a first molecular phylogeny based on cox2 sequences comprising all genera of the GDM currently accepted, with both lasting (Graminivora, Poakatesthia, and Viennotia) and evanescent (Peronosclerospora, Sclerophthora, and Sclerospora) sporangiophores. In addition, all other downy mildew genera currently accepted, as well as a representative sample of other oomycete taxa, have been included. It was shown that all genera of the GDM have had a long, independent evolutionary history, and that the delineation between Peronosclerospora and Sclerospora is correct. Sclerophthora was found to be a particularly divergent taxon nested within a paraphyletic Phytophthora, but without support. The results confirm that the placement of Peronosclerospora and Sclerospora in the Saprolegniomycetidae is incorrect. Sclerophthora is not closely related to Pachymetra of the family Verrucalvaceae, and also does not belong to the Saprolegniomycetidae, but shows close affinities to the Peronosporaceae. In addition, all GDM are interspersed throughout the Peronosporaceae s lat., suggesting that a separate family for the Sclerosporaceae might not be justified.

Comparison of sampling sites and detection methods for Haemophilus parasuis

Objective To improve the isolation rate and identification procedures for Haemophilus parasuis from pig tissues. Design Thirteen sampling sites and up to three methods were used to confirm the presence of H. parasuis in pigs after experimental challenge. Procedure Colostrum-deprived, naturally farrowed pigs were challenged intratracheally with H parasuis serovar 12 or 4. Samples taken during necropsy were either inoculated onto culture plates, processed directly for PCR or enriched prior to being processed for PCR. The recovery of H parasuis from different sampling sites and using different sampling methods was compared for each serovar. Results H parasuis was recovered from several sample sites for all serovar 12 challenged pigs, while the trachea was the only positive site for all pigs following serovar 4 challenge. The method of solid medium culture of swabs, and confirmation of the identity of cultured bacteria by PCR, resulted in 38% and 14% more positive results on a site basis for serovars 12 and 4, retrospectively, than direct PCR on the swabs. This difference was significant in the serovar 12 challenge. Conclusion Conventional culture proved to be more effective in detecting H parasuis than direct PCR or PCR on enrichment broths. For subacute (serovar 4) infections, the most successful sites for culture or direct PCR were pleural fluid, peritoneal fibrin and fluid, lung and pericardial fluid. For acute (serovar 12) infections, the best sites were lung, heart blood, affected joints and brain. The methodologies and key sampling sites identified in this study will enable improved isolation of H parasuis and aid the diagnosis of Glässer's disease.

Rapid isolation and detection of erythropoietin in blood plasma by magnetic core gold nanoparticles and portable Raman spectroscopy

Isolating, purifying, and identifying proteins in complex biological matrices is often difficult, time consuming, and unreliable. Herein we describe a rapid screening technique for proteins in biological matrices that combines selective protein isolation with direct surface enhanced Raman spectroscopy (SERS) detection. Magnetic core gold nanoparticles were synthesised, characterised, and subsequently functionalized with recombinant human erythropoietin (rHuEPO)-specific antibody. The functionalized nanoparticles were used to capture rHuEPO from horse blood plasma within 15 minutes. The selective binding between the protein and the functionalized nanoparticles was monitored by SERS. The purified protein was then released from the nanoparticles’ surface and directly spectroscopically identified on a commercial nanopillar SERS substrate. ELISA independently confirmed the SERS identification and quantified the released rHuEPO. Finally, the direct SERS detection of the extracted protein was successfully demonstrated for in-field screening by a handheld Raman spectrometer within 1 minute sample measurement time.

Rapid and sensitive detection of Avibacterium paragallinarum in the presence of other bacteria using a 5' Taq nuclease assay: A new tool for diagnosing infectious coryza

A 5' Taq nuclease assay specific for Avibacterium paragallinarum was designed and optimized for use in diagnosing infectious coryza. The region chosen for assay design was one of known specificity for Av. paragallinarum. The assay detected Av. paragallinarum reference strains representing the three Page and the eight Kume serovars, and field isolates from diverse geographical locations. No cross-reactions were observed with other Avibacterium species, with other bacteria taxonomically related to Av. paragallinarum nor with bacteria and viruses likely to be present in swabs collected from suspected infectious coryza cases. The detection limit for the assay was 6 to 60 colony-forming units per reaction. Twenty-two out of 53 swabs collected from sick birds reacted in the 5' Taq nuclease assay, whereas Av. paragallinarum was not isolated from any of the swabs. All of the 22 swabs yielded other bacteria in culture. The presence of Av. paragallinarum in the swabs was also demonstrated by sequencing, thereby confirming the ability of the assay to detect Av. paragallinarum in the presence of other bacteria. The ability to quantify bacterial load in the swabs using the 5' Taq nuclease assay was demonstrated.

The use of high resolution melting (HRM) to map single nucleotide polymorphism markers linked to a covered smut resistance gene in barley

Using an established genetic map, a single gene conditioning covered smut resistance, Ruh.7H, was mapped to the telomere region of chromosome 7HS in an Alexis/Sloop doubled haploid barley population. The closest marker to Ruh.7H, abg704 was 7.5 cM away. Thirteen loci on the distal end of 7HS with potential to contain single nucleotide polymorphisms (SNPs) were identified by applying a comparative genomics approach using rice sequence data. Of these, one locus produced polymorphic co-dominant bands of different size while two further loci contained SNPs that were identified using the recently developed high resolution melting (HRM) technique. Two of these markers flanked Ruh.7H with the proximal marker located 3.8 cM and the distal marker 2.7 cM away. This is the first report on the application of the HRM technique to SNP detection and to rapid scoring of known cleaved amplified polymorphic sequence (CAPS) markers in plants. This simple, precise post-PCR technique should find widespread use in the fine-mapping of genetic regions of interest in complex cereal and other plant genomes.

Sensory auditory processing and intuitive sound detection : an investigation of musical experts and nonexperts

The auditory system can detect occasional changes (deviants) in acoustic regularities without the need for subjects to focus their attention on the sound material. Deviant detection is reflected in the elicitation of the mismatch negativity component (MMN) of the event-related potentials. In the studies presented in this thesis, the MMN is used to investigate the auditory abilities for detecting similarities and regularities in sound streams. To investigate the limits of these processes, professional musicians have been tested in some of the studies. The results show that auditory grouping is already more advanced in musicians than in nonmusicians and that the auditory system of musicians can, unlike that of nonmusicians, detect a numerical regularity of always four tones in a series. These results suggest that sensory auditory processing in musicians is not only a fine tuning of universal abilities, but is also qualitatively more advanced than in nonmusicians. In addition, the relationship between the auditory change-detection function and perception is examined. It is shown that, contrary to the generally accepted view, MMN elicitation does not necessarily correlate with perception. The outcome of the auditory change-detection function can be implicit and the implicit knowledge of the sound structure can, after training, be utilized for behaviorally correct intuitive sound detection. These results illustrate the automatic character of the sensory change detection function.

Detection of nitrogen deficiency in wheat from spectral reflectance indices and basic crop eco-physiological concepts

We tested the capacity of several published multispectral indices to estimate the nitrogen nutrition of wheat canopies grown under different levels of water supply and plant density and derived a simple canopy reflectance index that is greatly independent of those factors. Planar domain geometry was used to account for mixed signals from the canopy and soil when the ground cover was low. A nitrogen stress index was developed, which adjusts shoot %N for plant biomass and area, thereby accounting for environmental conditions that affect growth, such as crop water status. The canopy chlorophyll content index (CCCi) and the modified spectral ratio planar index (mSRPi) could explain 68 and 69% of the observed variability in the nitrogen nutrition of the crop as early as Zadoks 33, irrespective of water status or ground cover. The CCCi was derived from the combination of 3 wavebands 670, 720 and 790 nm, and the mSRPi from 445, 705 and 750 nm, together with broader bands in the NIR and RED. The potential for their spatial application over large fields/paddocks is discussed.

Identification of two distinct bovine parainfluenza virus type 3 genotypes

The partial gene sequencing of the matrix (M) protein from seven clinical isolates of bovine parainfluenza virus type 3 (BPIV-3), and the complete sequencing of a representative isolate (Q5592) was completed in this study. Nucleotide sequence analysis was initiated because of the failure of in-house BPIV-3 RT-PCR methods to yield expected products for four of the isolates. Phylogenetic reconstructions based on the nucleotide sequences for the M-protein and the entire genome, using all of the available BPIV-3 nucleotide sequences, demonstrated that there were two distinct BPIV-3 genotypes (BPIV-3a and BPIV-3b). These newly identified genotypes have implications for the development of BPIV-3 molecular detection methods and may also impact on BPIV-3 vaccine formulations.