Comparison of Culture and a Novel 5' Taq Nuclease Assay for Direct Detection of Campylobacter fetus subsp. venerealis in Clinical Specimens from Cattle

A Campylobacter fetus subsp. venerealis-specific 5' Taq nuclease PCR assay using a 3' minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5' Taq nuclease assay detected approximately one single cell compared to 100 and 10 cells in the conventional PCR assay and 2,500 and 25,000 cells from selective culture from inoculated smegma and mucus, respectively. The respective detection limits following the enrichments from smegma and mucus were 5,000 and 50 cells/inoculum for the conventional PCR compared to 500 and 50 cells/inoculum for the 5' Taq nuclease assay. Field sampling confirmed the sensitivity and the specificity of the 5' Taq nuclease assay by detecting an additional 40 bulls that were not detected by culture. Urine-inoculated samples demonstrated comparable detection of C. fetus subsp. venerealis by both culture and the 5' Taq nuclease assay; however, urine was found to be less effective than smegma for bull sampling. Three infected bulls were tested repetitively to compare sampling tools, and the bull rasper proved to be the most suitable, as evidenced by the improved ease of specimen collection and the consistent detection of higher levels of C. fetus subsp. venerealis. The 5' Taq nuclease assay demonstrates a statistically significant association with culture (2 = 29.8; P < 0.001) and significant improvements for the detection of C. fetus subsp. venerealis-infected animals from crude clinical extracts following prolonged transport.

Production of microalgal concentrates by flocculation and their assessment as aquaculture feeds

A novel technique was developed for the flocculation of marine microalgae commonly used in aquaculture. The process entailed an adjustment of pH of culture to between 10 and 10.6 using NaOH, followed by addition of a non-ionic polymer Magnafloc LT-25 to a final concentration of 0.5 mg L-1. The ensuing flocculate was harvested, and neutralised giving a final concentration factor of between 200- and 800-fold. This process was successfully applied to harvest cells of Chaetoceros calcitrans, C. muelleri, Thalassiosira pseudonana, Attheya septentrionalis, Nitzschia closterium, Skeletonema sp., Tetraselmis suecica and Rhodomonas salina, with efficiencies >=80%. The process was rapid, simple and inexpensive, and relatively cost neutral with increasing volume (cf. concentration by centrifugation). Harvested material was readily disaggregated to single cell suspensions by dilution in seawater and mild agitation. Microscopic examination of the cells showed them to be indistinguishable from corresponding non-flocculated cells. Chlorophyll analysis of concentrates prepared from cultures of Concentrates of T. pseudonana prepared using pH-induced flocculation gave better growth of juvenile Pacific oysters (Crassostrea gigas) than concentrates prepared by ferric flocculation, or centrifuged concentrates using a cream separator or laboratory centrifuge. In follow up experiments, concentrates prepared from 1000 L Chaetoceros muelleri cultures were effective as supplementary diets to improve the growth of juvenile C. gigas and the scallop Pecten fumatus reared under commercial conditions, though not as effective as the corresponding live algae. The experiments demonstrated a proof-of-concept for a commercial application of concentrates prepared by flocculation, especially for use at a remote nursery without on-site mass-algal culture facilities.

A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits

Background: Molecular marker technologies are undergoing a transition from largely serial assays measuring DNA fragment sizes to hybridization-based technologies with high multiplexing levels. Diversity Arrays Technology (DArT) is a hybridization-based technology that is increasingly being adopted by barley researchers. There is a need to integrate the information generated by DArT with previous data produced with gel-based marker technologies. The goal of this study was to build a high-density consensus linkage map from the combined datasets of ten populations, most of which were simultaneously typed with DArT and Simple Sequence Repeat (SSR), Restriction Enzyme Fragment Polymorphism (RFLP) and/or Sequence Tagged Site (STS) markers. Results: The consensus map, built using a combination of JoinMap 3.0 software and several purpose-built perl scripts, comprised 2,935 loci (2,085 DArT, 850 other loci) and spanned 1,161 cM. It contained a total of 1,629 'bins' (unique loci), with an average inter-bin distance of 0.7 ± 1.0 cM (median = 0.3 cM). More than 98% of the map could be covered with a single DArT assay. The arrangement of loci was very similar to, and almost as optimal as, the arrangement of loci in component maps built for individual populations. The locus order of a synthetic map derived from merging the component maps without considering the segregation data was only slightly inferior. The distribution of loci along chromosomes indicated centromeric suppression of recombination in all chromosomes except 5H. DArT markers appeared to have a moderate tendency toward hypomethylated, gene-rich regions in distal chromosome areas. On the average, 14 ± 9 DArT loci were identified within 5 cM on either side of SSR, RFLP or STS loci previously identified as linked to agricultural traits. Conclusion: Our barley consensus map provides a framework for transferring genetic information between different marker systems and for deploying DArT markers in molecular breeding schemes. The study also highlights the need for improved software for building consensus maps from high-density segregation data of multiple populations.

Spirulina (Spirulina platensis) algae supplementation increases microbial protein production and feed intake and decreases retention time of digesta in the rumen of cattle

Cattle consuming pastures low in protein have low liveweight gain due to low rumen degradable protein (RDP) supply and thus low microbial crude protein (MCP) production and efficiency of MCP production [EMCP, g MCP/kg digestible organic matter (DOM)]. Nitrogen supplements can increase MCP production and EMCP of cattle grazing low protein pastures. The objective of this study was to compare the effects of supplementation with a non-protein-N source (NPN), in this case urea and ammonium sulfate (US), with a single-cell algal protein source (Spirulina platensis), on intake, microbial protein supply and digestibility in cattle. Nine cannulated Bos indicus steers [initial liveweight 250.1 ± 10.86 (s.d.) kg] were fed Mitchell grass hay (Astrebla spp; 6.1 g N, 746 g NDF/kg DM) ad libitum and were supplied with increasing amounts of US (0, 6, 13, 19 and 33 g US DM/kg hay DM) or Spirulina 0, 0.5, 1.4, 2.5 and 6.1 g Spirulina DM/kg W.day in an incomplete Latin square design. The response of MCP production and EMCP to increasing amounts of the two supplements was different, with a greater response to Spirulina evident. The MCP production was predicted to peak at 140 and 568 g MCP/day (0.64 and 2.02 g MCP/kg W.day) for the US and Spirulina supplements, respectively. The highest measured EMCP were 92 and 166 g MCP/kg DOM for the US and Spirulina treatments at 170 and 290 g RDP/kg DOM, respectively, or a Spirulina intake of 5.7 g DM/kg W.day. Increasing RDP intake from US and Spirulina resulted in an increase in Mitchell grass hay intake and rumen NH3-N concentration and reduced the retention time of liquid and particulate markers and digesta DM, NDF and lignin in the rumen with greater changes due to Spirulina. Total DM intake peaked at a Spirulina supplement level of 4.6 g Spirulina DM/kg W.day with a 2.3-fold higher DOM intake than Control steers. Rumen NH3-N concentrations reached 128 and 264 mg NH3-N/L for the US and Spirulina treatments with a significant increase in the concentration of branched-chain fatty acids for the Spirulina treatment. The minimum retention time of liquid (Cr-EDTA; 23 and 13 h) and particulate (Yb; 34 and 22 h) markers in the rumen were significantly lower for Spirulina compared with US and lower than unsupplemented animals at 24 and 34 h for Cr-EDTA and Yb, respectively. Spirulina could be provided safely at much higher N intakes than NPN supplements. The results suggest that, at an equivalent RDP supply, Spirulina provided greater increases than US in MCP production, EMCP and feed intake of Bos indicus cattle consuming low protein forage and could also be fed safely at higher levels of N intake.

Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy® or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to 1 infected in 800 samples with pepper but never detecting more than 1 infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

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.

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.

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.

An improved real-time PCR assay for the detection of Old World screwworm flies

The Old World screwworm (OWS) fly, Chrysomya bezziana, is a serious pest of livestock, wildlife and humans in tropical Africa, parts of the Middle East, the Indian subcontinent, south-east Asia and Papua New Guinea. Although to date Australia remains free of OWS flies, an incursion would have serious economic and animal welfare implications. For these reasons Australia has an OWS fly preparedness plan including OWS fly surveillance with fly traps. The recent development of an improved OWS fly trap and synthetic attractant and a specific and sensitive real-time PCR molecular assay for the detection of OWS flies in trap catches has improved Australia's OWS fly surveillance capabilities. Because all Australian trap samples gave negative results in the PCR assay, it was deemed necessary to include a positive control mechanism to ensure that fly DNA was being successfully extracted and amplified and to guard against false negative results. A new non-competitive internal amplification control (IAC) has been developed that can be used in conjunction with the OWS fly PCR assay in a multiplexed single-tube reaction. The multiplexed assay provides an indicator of the performance of DNA extraction and amplification without greatly increasing labour or reagent costs. The fly IAC targets a region of the ribosomal 16S mitochondrial DNA which is conserved across at least six genera of commonly trapped flies. Compared to the OWS fly assay alone, the multiplexed OWS fly and fly IAC assay displayed no loss in sensitivity or specificity for OWS fly detection. The multiplexed OWS fly and fly IAC assay provides greater confidence for trap catch samples returning negative OWS fly results. © 2014 International Atomic Energy Agency.