Detection of Chlamydiaceae DNA in veterinary specimens using a family-specific PCR

Aims: The aim of this work was to develop a rapid molecular test for the detection of the Chlamydiaceae family, irrespective of the species or animal host. Methods and Results: The method described herein is a polymerase chain reaction targeting the 16S rRNA gene of the Chlamydiaceae family, and the results demonstrate that the test reacts with five reference Chlamydiaceae but none of the 19 other bacterial species or five uninfected animal tissues tested. The results also indicate the enhanced sensitivity of this test when compared with conventional culture or serology techniques. This is demonstrated through parallel testing of six real clinical veterinary cases and confirmatory DNA sequence analysis. Conclusions, Significance and Impact of the Study: This test can be used by veterinary diagnostic laboratories for rapid detection of Chlamydiaceae in veterinary specimens, with no restriction of chlamydial species or animal host. The test does not differentiate chlamydial species, and if required, speciation must be carried out retrospectively using alternate methods. However, for the purpose of prescribing therapy for chlamydiosis, this test would be an invaluable laboratory tool.

Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Babesia bigemina Antibodies in Cattle

A competitive enzyme-linked immunosorbent assay (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C terminus of Babesia bigemina rhoptry-associated protein 1a was validated for international use. Receiver operating characteristic analysis revealed 16% inhibition as the threshold for a negative result, with an associated specificity of 98.3% and sensitivity of 94.7%. Increasing the threshold to 21% increased the specificity to 100% but modestly decreased the sensitivity to 87.2%. By using 21% inhibition, the positive predictive values ranged from 90.7% (10% prevalence) to 100% (95% prevalence) and the negative predictive values ranged from 97.0% (10% prevalence) to 48.2% (95% prevalence). The assay was able to detect serum antibody as early as 7 days after intravenous inoculation. The cELISA was distributed to five different laboratories along with a reference set of 100 defined bovine serum samples, including known positive, known negative, and field samples. The pairwise concordance among the five laboratories ranged from 100% to 97%, and all kappa values were above 0.8, indicating a high degree of reliability. Overall, the cELISA appears to have the attributes necessary for international application.

Detection of equine herpesvirus type 1 using a real-time polymerase chain reaction.

Equid herpesvirus 1 (EHV1) is a major disease of equids worldwide causing considerable losses to the horse industry. A variety of techniques, including PCR have been used to diagnose EHV1. Some of these PCRs were used in combination with other techniques such as restriction enzyme analysis (REA) or hybridisation, making them cumbersome for routine diagnostic testing and increasing the chances of cross-contamination. Furthermore, they involve the use of suspected carcinogens such as ethidium bromide and ultraviolet light. In this paper, we describe a real-time PCR, which uses minor groove-binding probe (MGB) technology for the diagnosis of EHV1. This technique does not require post-PCR manipulations thereby reducing the risk of cross-contamination. Most importantly, the technique is specific; it was able to differentiate EHV1 from the closely related member of the Alphaherpesvirinae, equid herpesvirus 4 (EHV4). It was not reactive with common opportunistic pathogens such as Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Enterobacter agglomerans often involved in abortion. Similarly, it did not react with equine pathogens such as Streptococcus equi, Streptococcus equisimilis, Streptococcus zooepidemicus, Taylorella equigenitalis and Rhodococcus equi, which also cause abortion. The results obtained with this technique agreed with results from published PCR methods. The assay was sensitive enough to detect EHV1 sequences in paraffin-embedded tissues and clinical samples. When compared to virus isolation, the test was more sensitive. This test will be useful for the routine diagnosis of EHV1 based on its specificity, sensitivity, ease of performance and rapidity.

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.

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**).

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.

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.

Trapping to monitor tephritid movement: Results, best practice, and assessment of alternatives

Movement of tephritid flies underpins their survival, reproduction, and ability to establish in new areas and is thus of importance when designing effective management strategies. Much of the knowledge currently available on tephritid movement throughout landscapes comes from the use of direct or indirect methods that rely on the trapping of individuals. Here, we review published experimental designs and methods from mark-release-recapture (MRR) studies, as well as other methods, that have been used to estimate movement of the four major tephritid pest genera (Bactrocera, Ceratitis, Anastrepha, and Rhagoletis). In doing so, we aim to illustrate the theoretical and practical considerations needed to study tephritid movement. MRR studies make use of traps to directly estimate the distance that tephritid species can move within a generation and to evaluate the ecological and physiological factors that influence dispersal patterns. MRR studies, however, require careful planning to ensure that the results obtained are not biased by the methods employed, including marking methods, trap properties, trap spacing, and spatial extent of the trapping array. Despite these obstacles, MRR remains a powerful tool for determining tephritid movement, with data particularly required for understudied species that affect developing countries. To ensure that future MRR studies are successful, we suggest that site selection be carefully considered and sufficient resources be allocated to achieve optimal spacing and placement of traps in line with the stated aims of each study. An alternative to MRR is to make use of indirect methods for determining movement, or more correctly, gene flow, which have become widely available with the development of molecular tools. Key to these methods is the trapping and sequencing of a suitable number of individuals to represent the genetic diversity of the sampled population and investigate population structuring using nuclear genomic markers or non-recombinant mitochondrial DNA markers. Microsatellites are currently the preferred marker for detecting recent population displacement and provide genetic information that may be used in assignment tests for the direct determination of contemporary movement. Neither MRR nor molecular methods, however, are able to monitor fine-scale movements of individual flies. Recent developments in the miniaturization of electronics offer the tantalising possibility to track individual movements of insects using harmonic radar. Computer vision and radio frequency identification tags may also permit the tracking of fine-scale movements by tephritid flies by automated resampling, although these methods come with the same problems as traditional traps used in MRR studies. Although all methods described in this chapter have limitations, a better understanding of tephritid movement far outweighs the drawbacks of the individual methods because of the need for this information to manage tephritid populations.

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.

Confirmation of QTL mapping and marker validation for partial seedling resistance to crown rot in wheat line '2-49'

We have tested the efficacy of putative microsatellite single sequence repeat (SSR) markers, previously identified in a 2-49 (Gluyas Early/Gala) × Janz doubled haploid wheat (Triticum aestivum) population, as being linked to partial seedling resistance to crown rot disease caused by Fusarium pseudograminearum. The quantitative trait loci (QTLs) delineated by these markers have been tested for linkage to resistance in an independent Gluyas Early × Janz doubled haploid population. The presence of a major QTL on chromosome 1DL (QCr.usq-1D1) and a minor QTL on chromosome 2BS (QCr.usq-2B1) was confirmed. However, a putative minor QTL on chromosome 2A was not confirmed. The QTL on 1D was inherited from Gluyas Early, a direct parent of 2-49, whereas the 2B QTL was inherited from Janz. Three other putative QTLs identified in 2-49 × Janz (on 1AL, 4BL, and 7BS) were inherited by 2-49 from Gala and were not able to be confirmed in this study. The screening of SSR markers on a small sample of elite wheat genotypes indicated that not all of the most tightly linked SSR markers flanking the major QTLs on 1D and 1A were polymorphic in all backgrounds, indicating the need for additional flanking markers when backcrossing into some elite pedigrees. Comparison of SSR haplotypes with those of other genotypes exhibiting partial crown rot resistance suggests that additional, novel sources of crown rot resistance are available.

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.