Comparative distribution of eukaryotic sequences in Great Barrier Reef coral holobionts.

<p>(A) Abundance of sequences belonging taxonomic groups of Eukaryotes in corals using three tag encoded 454 amplicon pyrosequencing assays targeting eukaryote small-subunit rRNA gene assays (Euk1, Euk2, Euk3). Relative position of the universal primer pairs utilised Euk1-3 assays is schematically shown on a complete small-subunit rRNA gene (inset right). (B) Venn diagram of sequence 97% sequence identity clusters shared between corals according to Euk1-3 assays. Number of sequences used to calculate Venn diagram within 97% clusters (>1 sequence) obtained using Euk1-3 assay and DNA indicated on the left (colour coded according to coral DNA). Total number of sequences recovered for each Euk1-3 (number in brackets on the right) and number sequences represented by >1 sequence (n). Coral species <i>Acroporapalifera</i> 42a, <i>Montipora</i><i>digitata</i> 60a, <i>Porites</i><i>cylindrica</i> 66-2 from Heron Island and <i>Seriatopora</i><i>hystrix</i> 176-2 from the One Tree Island.</p

Phylogenetic tree of apicomplexan N-symbiont from the Great Barrier Reef based on small-subunit rRNA gene sequences.

<p>The tree was inferred using the Maximum Likelihood method based on the Tamura-Nei model with a discrete Gamma distribution (5 categories, alpha parameter = 0.2444). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 30 nucleotide sequences and rooted with <i>Cryptosporidium</i> spp. sequences. GenBank accession is shown on the right of the species name. Bootstrap support inferred from 100 replicates is shown (>50%). All positions with less than 75% site coverage were eliminated. There were a total of 1463 positions in the final dataset. Evolutionary analyses were conducted in MEGA5.1.</p

Distribution of bacterial sequences in Great Barrier Reef coral holobionts.

<p>(A) Rarefaction curves with 95% confidence interval for sequences obtained using bacterial tag encoded 454 amplicon pyrosequencing assays targeting small-subunit rRNA gene assays (Bac1). Dominance and Chao-1 are indicated above each graph. (B) Abundance of sequences belonging bacterial phyla. Proportion of sequences belonging to bacterial classes within phylum Proteobacteria (inset). Coral species <i>Acroporapalifera</i> 42a, <i>Montipora</i><i>digitata</i> 60a, <i>Porites</i><i>cylindrica</i> 66-2 from Heron Island and <i>Seriatopora</i><i>hystrix</i> 176-2 from the One Tree Island.</p

Combined Amplicon Pyrosequencing Assays Reveal Presence of the Apicomplexan “type-N” (cf. <i>Gemmocystis cylindrus</i>) and <i>Chromera</i><i> velia</i> on the Great Barrier Reef, Australia

<div><p>Background</p><p>The coral is predominantly composed of the metabolically dependent coral host and the photosynthetic dinoflagellate <i>Symbiodinium</i> sp. The system as a whole interacts with symbiotic eukaryotes, bacteria and viruses. <i>Gemmocystiscylindrus</i> (cf. “type-N” symbiont) belonging to the obligatory parasitic phylum Apicomplexa (Alveolata) is ubiquitous in the Caribbean coral, but its presence in the Great Barrier Reef coral has yet to be documented. Approaches allowing identification of the healthy community from the pathogenic or saprobic organisms are needed for sustainable coral reef monitoring.</p> <p>Methods & Principal Findings</p><p>We investigated the diversity of eukaryotes associated with a common reef-building corals from the southern Great Barrier Reef. We used three tag encoded 454 amplicon pyrosequencing assays targeting eukaryote small-subunit rRNA gene to demonstrate the presence of the apicomplexan type-N and a photosynthetic sister species to Apicomplexa - <i>Chromera</i><i>velia</i>. Amplicon pyrosequencing revealed presence of the small-subunit rRNA genes of known eukaryotic pathogens (<i>Cryptosporidium</i> and <i>Cryptococcus</i>). We therefore conducted bacterial tag encoded amplicon pyrosequencing assay for small-subunit rRNA gene to support effluent exposure of the coral. Bacteria of faecal origin (Enterobacteriales) formed 41% of total sequences in contrast to 0-2% of the coral-associated bacterial communities with and without <i>C</i><i>. velia</i>, respectively.</p> <p>Significance</p><p>This is the first time apicomplexan type-N has been detected in the Great Barrier Reef. Eukaryote tag encoded amplicon pyrosequencing assays demonstrate presence of apicomplexan type-N and <i>C. Velia</i> in total coral DNA. The data highlight the need for combined approaches for eukaryotic diversity studies coupled with bacterial community assessment to achieve a more realistic goals of defining the holobiont community and assessing coral disease. With increasing evidence of Apicomplexa in coral reef environments, it is important not only to understand the evolution of these organisms but also identify their potential as pathogens.</p> </div

Correlation between faecal egg counts and real-time PCR egg estimates.

<p>(A) Real-time PCR egg estimates (qEPG) for three methods of sample preparation for DNA isolation from cattle samples collected during Austral spring (Herd 1) are compared to morphological FEC (mEPG); isolation of DNA from 150 mg raw faeces (Method 1), sedimentation followed by isolation of DNA from 150 μl of the resultant sediment (Method 2), and sedimentation followed by isolation of DNA from the entire sediment pellet (Method 3). (B) As for A using only Method 3 on samples collected during Austral summer (Herd 1). (C) As for B using samples collected from an endemic population (Herd 2). (D) Correlation between duplicate sedimentations (mEPG) for all samples (Herds 1 and 2) across all sampling periods; inset–correlation between duplicate FECs for samples with ≤ 20 EPG.</p

C_T values of 2000 clean <i>F</i>. <i>hepatica</i> eggs in phosphate buffered saline (PBS) when subjected to six disruption treatments.

<p>C_T values of 2000 clean <i>F</i>. <i>hepatica</i> eggs in phosphate buffered saline (PBS) when subjected to six disruption treatments.</p

Correlation between faecal egg counts and real-time PCR egg estimates.

<p>(A) Real-time PCR egg estimates (qEPG) for three methods of sample preparation for DNA isolation from cattle samples collected during Austral spring (Herd 1) are compared to morphological FEC (mEPG); isolation of DNA from 150 mg raw faeces (Method 1), sedimentation followed by isolation of DNA from 150 μl of the resultant sediment (Method 2), and sedimentation followed by isolation of DNA from the entire sediment pellet (Method 3). (B) As for A using only Method 3 on samples collected during Austral summer (Herd 1). (C) As for B using samples collected from an endemic population (Herd 2). (D) Correlation between duplicate sedimentations (mEPG) for all samples (Herds 1 and 2) across all sampling periods; inset–correlation between duplicate FECs for samples with ≤ 20 EPG.</p

Scrambled eggs: A highly sensitive molecular diagnostic workflow for <i>Fasciola</i> species specific detection from faecal samples

<div><p>Background</p><p>Fasciolosis, due to <i>Fasciola hepatica</i> and <i>Fasciola gigantica</i>, is a re-emerging zoonotic parasitic disease of worldwide importance. Human and animal infections are commonly diagnosed by the traditional sedimentation and faecal egg-counting technique. However, this technique is time-consuming and prone to sensitivity errors when a large number of samples must be processed or if the operator lacks sufficient experience. Additionally, diagnosis can only be made once the 12-week pre-patent period has passed. Recently, a commercially available coprological antigen ELISA has enabled detection of <i>F</i>. <i>hepatica</i> prior to the completion of the pre-patent period, providing earlier diagnosis and increased throughput, although species differentiation is not possible in areas of parasite sympatry. Real-time PCR offers the combined benefits of highly sensitive species differentiation for medium to large sample sizes. However, no molecular diagnostic workflow currently exists for the identification of <i>Fasciola</i> spp. in faecal samples.</p><p>Methodology/Principal findings</p><p>A new molecular diagnostic workflow for the highly-sensitive detection and quantification of <i>Fasciola</i> spp. in faecal samples was developed. The technique involves sedimenting and pelleting the samples prior to DNA isolation in order to concentrate the eggs, followed by disruption by bead-beating in a benchtop homogeniser to ensure access to DNA. Although both the new molecular workflow and the traditional sedimentation technique were sensitive and specific, the new molecular workflow enabled faster sample throughput in medium to large epidemiological studies, and provided the additional benefit of speciation. Further, good correlation (R² = 0.74–0.76) was observed between the real-time PCR values and the faecal egg count (FEC) using the new molecular workflow for all herds and sampling periods. Finally, no effect of storage in 70% ethanol was detected on sedimentation and DNA isolation outcomes; enabling transport of samples from endemic to non-endemic countries without the requirement of a complete cold chain. The commercially-available ELISA displayed poorer sensitivity, even after adjustment of the positive threshold (65–88%), compared to the sensitivity (91–100%) of the new molecular diagnostic workflow.</p><p>Conclusions/Significance</p><p>Species-specific assays for sensitive detection of <i>Fasciola</i> spp. enable ante-mortem diagnosis in both human and animal settings. This includes Southeast Asia where there are potentially many undocumented human cases and where post-mortem examination of production animals can be difficult. The new molecular workflow provides a sensitive and quantitative diagnostic approach for the rapid testing of medium to large sample sizes, potentially superseding the traditional sedimentation and FEC technique and enabling surveillance programs in locations where animal and human health funding is limited.</p></div

Severe reactions to <i>Cystodiscus axonis</i> plasmodia in other species of Australian frogs.

<p>A. Spinal cord of Southern bell frog showing multifocal and locally extensive gliosis in reaction to moderate load of <i>C. axonis</i> plasmodia (arrows), individual was noted to be lethargic and inappetent, scale 200 µm; B. Spinal cord of Southern bell frog showing congestion and severe haemorrhage in association with high numbers of <i>C. axonis</i> plasmodia, individual was found dead, scale 50 µm; C. Diffuse haemorrhage, multifocal melacia (grey arrowheads) and gliosis and <i>C. axonis</i> plasmodia (black arrowheads) in spinal cord of Yellow spotted bell frog, scale 100 µm; D. <i>C. axonis</i> plasmodia associated with blood vessel epithelial cell in Yellow spotted bell frog spinal cord, scale 20 µm; E. Cross section of Booroolong frog infected with <i>C. axonis</i>, showing severe haemorrhage, extensive multifocal gliosis and spongiform change, scale 100 µm; F. Nerve root of Striped marsh frog showing mild focal gliosis in association with <i>C. axonis</i> plasmodia (arrow), scale 50 µm.</p

C_T values of <i>F</i>. <i>hepatica</i> eggs in sheep faecal samples when subjected to three different disruption treatments.

<p>C_T values of <i>F</i>. <i>hepatica</i> eggs in sheep faecal samples when subjected to three different disruption treatments.</p