Publications by authors named "Andrew C Barnes"

53 Publications

Complete, closed and curated genome sequences of subsp. isolates from Australia indicate mobilome-driven localized evolution and novel pathogenicity determinants.

Microb Genom 2021 Apr;7(4)

School of Biological Sciences, University of Queensland, Brisbane, Queensland 4072, Australia.

Despite the recent advances in sequencing technologies, the complete assembly of multi-chromosome genomes of the , often containing several plasmids, remains challenging. Using a combination of Oxford Nanopore MinION long reads and short Illumina reads, we fully sequenced, closed and curated the genomes of two strains of a primary aquatic pathogen subsp. isolated in Australia. These are also the first genome sequences of subsp. isolated in Oceania and, to our knowledge, in the Southern hemisphere. We also investigated the phylogenetic relationships between Australian and overseas isolates, revealing that Australian subsp. are more closely related to the Asian and American strains rather than to the European ones. We investigated the mobilome and present new evidence showing that a host specialization process and progressive adaptive evolution to fish are ongoing in subsp. , and are largely mediated by transposable elements, predominantly in chromosome 2, and by plasmids. Finally, we identified two novel potential virulence determinants in subsp. - a chorismate mutase gene, which is ubiquitously retained and co-localized with the AIP56 apoptogenic toxin-encoding gene on the pPHDP10 plasmid, and transfer-messenger RNA gene located on the main chromosome, homologous to a critical-to-virulence determinant in . Our study describes, to our knowledge, the only fully closed and manually curated genomes of subsp. available to date, offering new insights into this important fish pathogen and its evolution.
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http://dx.doi.org/10.1099/mgen.0.000562DOI Listing
April 2021

Serum IgM heavy chain sub-isotypes and light chain variants revealed in giant grouper (Epinephelus lanceolatus) via protein A affinity purification, mass spectrometry and genome sequencing.

Fish Shellfish Immunol 2021 Jun 29;113:42-50. Epub 2021 Mar 29.

The University of Queensland, School of Biological Sciences, Australia. Electronic address:

Two IgM heavy (H) chain sub-isotypes (80 and 40 kDa) and two light (L) chain variants (25 and 30 kDa) were detected in the serum of giant grouper (Epinephelus lanceolatus), purified by ammonium sulphate precipitation followed by protein A affinity chromatography. This method yielded 5.6 mg/mL high purity IgM from grouper serum, with efficiency estimated at 39.5% recovery from crude serum. The H and L chains were identified by SDS-PAGE and mass spectrometry (MS). Nanopore long-read sequencing was used to generate a genomic contig (MW768935), containing Cμ, Cδ loci, V regions, and a H chain Joining segment. cDNA sequencing of Cμ transcripts (MW768933 and MW768934) were used to polish the genomic contig and determine the exons and introns of the corresponding locus. MS peptide mapping revealed that the 80 kDa H chain consisted of C1-4 domains while peptides from the 40 kDa H chain only mapped to C1-2 domains. Our genomic contig showed the Cμ locus has a Cμ1-Cμ2-Cμ3-Cμ4 arrangement on the same strand as the other Ig loci identified in this genomic sequence. Our study corrects the NCBI annotations of the opposing Cμ loci (LOC117268697 and LOC117268550) in chromosome 16 (NC_047006). Further, we identified both κ and λ L chain isotypes in serum IgM. The molecular weight differences observed may result from different combinations of C and V genes. Putative IgM sub-isotypes have also been reported in Epinephelus itajara and Epinephelus coioides. The presence of IgM sub-isotypes may be a conserved trait among Epinephelus species.
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http://dx.doi.org/10.1016/j.fsi.2021.03.014DOI Listing
June 2021

Immersion challenge of naïve Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. did not increase the severity of Neoparamoeba perurans-induced amoebic gill disease (AGD).

J Fish Dis 2021 Feb 12;44(2):149-160. Epub 2020 Dec 12.

Livestock & Aquaculture, CSIRO, Queensland Biosciences Precinct, Brisbane, Qld, Australia.

Amoebic gill disease (AGD) is one of the main health issues impacting farmed Atlantic salmon. Neoparamoeba perurans causes AGD; however, a diversity of other amoeba species colonizes the gills and there is little understanding of whether they are commensal or potentially involved in different stages of gill disease development. Here, we conduct in vivo challenges of naïve Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. to investigate their pathogenicity to Atlantic salmon gills. Additionally, we assessed whether the presence of Nolandella sp. and Pseudoparamoeba sp. influences the onset and/or severity of N. perurans-induced AGD. All three strains attached and multiplied on the gills according to qPCR analysis. Furthermore, minor gross gill lesions and histological changes were observed post-exposure. While N. perurans was found associated with classical AGD lesions, Nolandella sp. and Pseudoparamoeba sp. were not found associated with lesion sites and these lesions did not meet the expected composite of histopathological changes for AGD. Moreover, the presence of these non-N. perurans species did not significantly increase the severity of AGD. This trial provides evidence that cultured Nolandella sp. and Pseudoparamoeba sp. do not induce AGD and do not influence the severity of AGD during the early stages of development.
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http://dx.doi.org/10.1111/jfd.13319DOI Listing
February 2021

Evolutionary epidemiology of Streptococcus iniae: Linking mutation rate dynamics with adaptation to novel immunological landscapes.

Infect Genet Evol 2020 11 19;85:104435. Epub 2020 Jun 19.

The University of Queensland, School of Biological Sciences, St Lucia Campus, Brisbane, Queensland 4072, Australia. Electronic address:

Pathogens continuously adapt to changing host environments where variation in their virulence and antigenicity is critical to their long-term evolutionary success. The emergence of novel variants is accelerated in microbial mutator strains (mutators) deficient in DNA repair genes, most often from mismatch repair and oxidized-guanine repair systems (MMR and OG respectively). Bacterial MMR/OG mutants are abundant in clinical samples and show increased adaptive potential in experimental infection models, yet the role of mutators in the epidemiology and evolution of infectious disease is not well understood. Here we investigated the role of mutation rate dynamics in the evolution of a broad host range pathogen, Streptococcus iniae, using a set of 80 strains isolated globally over 40 years. We have resolved phylogenetic relationships using non-recombinant core genome variants, measured in vivo mutation rates by fluctuation analysis, identified variation in major MMR/OG genes and their regulatory regions, and phenotyped the major traits determining virulence in streptococci. We found that both mutation rate and MMR/OG genotype are remarkably conserved within phylogenetic clades but significantly differ between major phylogenetic lineages. Further, variation in MMR/OG loci correlates with occurrence of atypical virulence-associated phenotypes, infection in atypical hosts (mammals), and atypical (osseous) tissue of a vaccinated primary host. These findings suggest that mutators are likely to facilitate adaptations preceding major diversification events and may promote emergence of variation permitting colonization of a novel host tissue, novel host taxa (host jumps), and immune-escape in the vaccinated host.
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http://dx.doi.org/10.1016/j.meegid.2020.104435DOI Listing
November 2020

Antibiotic Use by Small-Scale Farmers for Freshwater Aquaculture in the Upper Mekong Delta, Vietnam.

J Aquat Anim Health 2019 09 11;31(3):290-298. Epub 2019 Sep 11.

Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Post Office Box 7036, 750 07, Uppsala, Sweden.

This study describes antibiotic use by small-scale freshwater aquaculture farmers in the upper Mekong Delta in southwestern Vietnam and the knowledge and practices surrounding the cause and prevention of aquaculture disease in that region. Forty five farmers were included in the study, of which 19 (42%) cultivated tilapia Oreochromis spp., 13 (29%) Striped Catfish Pangasianodon hypophthalmus and 13 (29%) giant river prawns Macrobrachium rosenbergii. Antibiotics were used by farmers of tilapia and Striped Catfish (84% and 69% of farmers, respectively), but not by any of the prawn farmers. Most farmers (72%) used antibiotics for around 3 d when treating diseases, depending on the farmers' economic means and whether the fish recovered, as judged by the farmer. If farmers perceived that the antibiotic treatment had failed, the most common response was to change to another type of antibiotic. Some farmers also used antibiotics in the absence of clinical symptoms as a preventive measure. In the absence of rapid, cost-effective diagnostics, the likelihood for the incorrect use of antibiotics is high, which has implications for antibiotic resistance. Moreover, the sequential use of different antibiotics following therapeutic failure is a risk factor for the emergence of resistance. All farmers that were surveyed were aware of the risks associated with antibiotic use. This may lead to successful intervention toward reduced antibiotic use in freshwater fish farming in Vietnam.
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http://dx.doi.org/10.1002/aah.10084DOI Listing
September 2019

Mixed culture purple phototrophic bacteria is an effective fishmeal replacement in aquaculture.

Water Res X 2019 Aug 24;4:100031. Epub 2019 Apr 24.

Advanced Water Management Centree, The University of Queensland, Brisbane, Queensland, 4072, Australia.

Aquaculture is the fastest growing animal food production industry, now producing 50% of all food fish. However, aquaculture feeds remain dependent on fishmeal derived from capture fisheries, which must be reduced for continued sustainable growth. Purple phototrophic bacteria (PPB) efficiently yield biomass from wastewater with high product homogeneity, a relatively high protein fraction, and potential added value as an ingredient for fish feeds. Here we test bulk replacement of fishmeal with PPB microbial biomass in diets for Asian sea bass (), a high value carnivorous fish with high protein to energy requirement. Mixed culture PPB were grown in a novel 1 m attached photo-biofilm process using synthetic and real wastewater. Four experimental diets were formulated to commercial specifications but with the fishmeal substituted (0%, 33%, 66%, and 100%) with the synthetic grown PPB biomass and fed to a cohort of 540 juvenile fish divided amongst 12 tanks over 47 days. Weight and standard length were taken from individual fish at 18, 28, and 47d. No significant difference in survival was observed due to diet or other factors (94-100%). There was a negative correlation between PPB inclusion level and final weight ( = 5.94 × 10) with diet accounting for 4.1% of the variance over the trial (general linear model, R = 0.96,  = 1 × 10). Feed conversion ratio was also significantly influenced by diet ( = 6 × 10) with this factor accounting for 89% of variance. Specifically, feed conversion ratio (FCR) rose to 1.5 for the 100% replacement diet during the last sample period, approximately 1.0 for the partial replacement, and 0.8 for the nil replacement diet. However, this study demonstrates that bulk replacement of fishmeal by PPB is feasible, and commercially viable at 33% and 66% replacement.
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http://dx.doi.org/10.1016/j.wroa.2019.100031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614599PMC
August 2019

Leucocyte integrins, but neither caspases nor NLR inflammasome are associated with lipopolysaccharide recognition and response in barramundi (Lates calcarifer).

Fish Shellfish Immunol 2019 Aug 16;91:172-179. Epub 2019 May 16.

School of Biological Sciences and Centre for Marine Science, The University of Queensland, St. Lucia, QLD, 4072, Australia. Electronic address:

The inflammatory response of fish to LPS is subdued, attributed to absence of TLR4, a key pro-inflammatory receptor for LPS in mammals. Nevertheless, LPS is processed in fish in a T-independent manner and is a protective antigen in fish vaccines, yet pathways for processing LPS in fish remain to be elucidated. Here, we report that caspases and NOD-like receptor inflammasomes typically responsible for LPS recognition and processing in mammals lack critical domains or are absent in barramundi (Lates calcarifer). On the other hand, leucocyte integrins MAC-1 and LFA-1 were detected on the surface of neutrophil- and lymphocyte-like cells respectively in the barramundi spleen by immunocytochemistry, and leucocytes displaying MAC-1 or LFA-1 bound to Factor X and ESM-1 respectively. Exposure to MAC-1 and LFA-1 induced significant IL-1β expression post-stimulation with LPS compared to unstimulated and isotype controls, but the differences observed in TNF-α expression were inconclusive. Our findings implicate MAC-1 and LFA-1 involvement in immune processing of LPS in barramundi and in antigen processing in fish.
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http://dx.doi.org/10.1016/j.fsi.2019.05.015DOI Listing
August 2019

Interactions of head-kidney leucocytes from giant grouper, Epinephelus lanceolatus, with pathogenic Streptococcus agalactiae strains from marine and terrestrial origins.

Fish Shellfish Immunol 2019 Jul 23;90:250-263. Epub 2019 Apr 23.

The University of Queensland, School of Biological Sciences and Centre for Marine Science, Brisbane, Queensland, 4072, Australia. Electronic address:

Streptococcus agalactiae (Group B Streptococcus, GBS) is emerging as a genetically diverse species infecting farmed and wild fish, including commercially and culturally important groupers. To better understand how S. agalactiae are pathogenic in fish, we investigated interactions between isolates from fish and terrestrial hosts and the cellular immune system of Queensland grouper Epinephelus lanceolatus using flow cytometry. Adherent head-kidney leucocytes (HKL) from Queensland grouper displayed two main cell populations with distinct forward and side scatter by flow cytometry. The population of smaller and less complex cells (P1) was composed of monocytes, lymphocytes and thrombocytes, while the population of primarily larger and more complex cells (P2) comprised predominantly of macrophages and neutrophils. The cells in P2 had higher phagocytic index and capacity when incubated with fluorescent latex beads. HKL were activated by phorbol myristate acetate (PMA) but were unresponsive to lipopolysaccharide (LPS) and peptidoglycan (PTG), suggesting the absence of specific receptors on the surface of these cells for these ligands or a requirement for intermediates. In in vitro phagocytosis assays, all fish isolates of GBS activated a respiratory burst in P2 indicated by significant production of intracellular reactive oxygen species (ROS). Similarly, dog and cat isolates of different serotype and sequence type also induced ROS production in grouper HKL. However, human, crocodile and bovine isolates of GBS did not elicit significant ROS in HKL although they coincided with the highest phagocytic index. This suggests that these strains are capable of quenching ROS production. Terrestrial isolates significantly increased mortality of Queensland grouper leucocytes in vitro, aligned with a more diverse repertoire of cellular toxins in these strains. Opsonisation of a marine strain and terrestrial strain of GBS with antiserum raised against the marine strain resulted in an increase in ROS production by HKL in both cases although there was low antigenic cross reactivity between the two strains by flow cytometry, reflecting their diverse serotypes (Ib vs III). However, pre-incubation of either strain with normal serum from grouper also increased ROS production of HKL suggesting other opsonins may be involved. Based on these results it appears that piscine and terrestrial GBS isolates have contrasting strategies when interacting with the cellular immune system of Queensland grouper; the former seemingly evading phagocytosis, whilst the latter are readily phagocytosed but counteract ROS production.
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http://dx.doi.org/10.1016/j.fsi.2019.04.058DOI Listing
July 2019

A diversity of amoebae colonise the gills of farmed Atlantic salmon (Salmo salar) with amoebic gill disease (AGD).

Eur J Protistol 2019 Feb 29;67:27-45. Epub 2018 Oct 29.

CSIRO Agriculture and Food, Integrated Sustainable Aquaculture Production, Queensland Biosciences Precinct, 306 Carmody Road, Brisbane, Queensland 4067, Australia.

Neoparamoeba perurans is the aetiological agent of amoebic gill disease (AGD) in salmonids, however multiple other amoeba species colonise the gills and their role in AGD is unknown. Taxonomic assessments of these accompanying amoebae on AGD-affected salmon have previously been based on gross morphology alone. The aim of the present study was to document the diversity of amoebae colonising the gills of AGD-affected farmed Atlantic salmon using a combination of morphological and sequence-based taxonomic methods. Amoebae were characterised morphologically via light microscopy and transmission electron microscopy, and by phylogenetic analyses based on the 18S rRNA gene and cytochrome oxidase subunit I (COI) gene. In addition to N. perurans, 11 other amoebozoans were isolated from the gills, and were classified within the genera Neoparamoeba, Paramoeba, Vexillifera, Pseudoparamoeba, Vannella and Nolandella. In some cases, such as Paramoeba eilhardi, this is the first time this species has been isolated from the gills of teleost fish. Furthermore, sequencing of both the 18S rRNA and COI gene revealed significant genetic variation within genera. We highlight that there is a far greater diversity of amoebae colonising AGD-affected gills than previously established.
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http://dx.doi.org/10.1016/j.ejop.2018.10.003DOI Listing
February 2019

Microevolution of Streptococcus agalactiae ST-261 from Australia Indicates Dissemination via Imported Tilapia and Ongoing Adaptation to Marine Hosts or Environment.

Appl Environ Microbiol 2018 08 1;84(16). Epub 2018 Aug 1.

The University of Queensland, School of Biological Sciences and Centre for Marine Science, St. Lucia Campus, Brisbane, Queensland, Australia

(group B [GBS]) causes disease in a wide range of animals. The serotype Ib lineage is highly adapted to aquatic hosts, exhibiting substantial genome reduction compared with terrestrial conspecifics. Here, we sequence genomes from 40 GBS isolates, including 25 isolates from wild fish and captive stingrays in Australia, six local veterinary or human clinical isolates, and nine isolates from farmed tilapia in Honduras, and compared them with 42 genomes from public databases. Phylogenetic analysis based on nonrecombinant core-genome single nucleotide polymorphisms (SNPs) indicated that aquatic serotype Ib isolates from Queensland were distantly related to local veterinary and human clinical isolates. In contrast, Australian aquatic isolates are most closely related to a tilapia isolate from Israel, differing by only 63 core-genome SNPs. A consensus minimum spanning tree based on core-genome SNPs indicates the dissemination of sequence type 261 (ST-261) from an ancestral tilapia strain, which is congruent with several introductions of tilapia into Australia from Israel during the 1970s and 1980s. Pangenome analysis identified 1,440 genes as core, with the majority being dispensable or strain specific, with non-protein-coding intergenic regions (IGRs) divided among core and strain-specific genes. Aquatic serotype Ib strains have lost many virulence factors during adaptation, but six adhesins were well conserved across the aquatic isolates and might be critical for virulence in fish and for targets in vaccine development. The close relationship among recent ST-261 isolates from Ghana, the United States, and China with the Israeli tilapia isolate from 1988 implicates the global trade in tilapia seed for aquaculture in the widespread dissemination of serotype Ib fish-adapted GBS. (GBS) is a significant pathogen of humans and animals. Some lineages have become adapted to particular hosts, and serotype Ib is highly specialized to fish. Here, we show that this lineage is likely to have been distributed widely by the global trade in tilapia for aquaculture, with probable introduction into Australia in the 1970s and subsequent dissemination in wild fish populations. We report here the variability in the polysaccharide capsule among this lineage but identify a cohort of common surface proteins that may be a focus of future vaccine development to reduce the biosecurity risk in international fish trade.
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http://dx.doi.org/10.1128/AEM.00859-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070751PMC
August 2018

Multilocus Variable-Number Tandem-Repeat Analysis of Yersinia ruckeri Confirms the Existence of Host Specificity, Geographic Endemism, and Anthropogenic Dissemination of Virulent Clones.

Appl Environ Microbiol 2018 08 1;84(16). Epub 2018 Aug 1.

Norwegian Veterinary Institute, Oslo, Norway

A multilocus variable-number tandem-repeat analysis (MLVA) assay was developed for epizootiological study of the internationally significant fish pathogen , which causes yersiniosis in salmonids. The assay involves amplification of 10 variable-number tandem-repeat (VNTR) loci in two five-plex PCRs, followed by capillary electrophoresis. A collection of 484 isolates, originating from various biological sources and collected from four continents over 7 decades, was analyzed. Minimum-spanning-tree cluster analysis of MLVA profiles separated the studied population into nine major clonal complexes and a number of minor clusters and singletons. The major clonal complexes could be associated with host species, geographic origin, and serotype. A single large clonal complex of serotype O1 isolates dominating the yersiniosis situation in international rainbow trout farming suggests anthropogenic spread of this clone, possibly related to transport of fish. Moreover, subclustering within this clonal complex indicates putative transmission routes and multiple biotype shift events. In contrast to the situation in rainbow trout, strains associated with disease in Atlantic salmon appear as more or less geographically isolated clonal complexes. A single complex of serotype O1 exclusive to Norway was found to be responsible for almost all major yersiniosis outbreaks in modern Norwegian salmon farming, and site-specific subclustering further indicates persistent colonization of freshwater farms in Norway. Identification of genetically diverse isolates from clinically healthy fish and environmental sources also suggests the widespread existence of less-virulent or avirulent strains. This comprehensive population study substantially improves our understanding of the epizootiological history and nature of an internationally important fish-pathogenic bacterium. The MLVA assay developed and presented represents a high-resolution typing tool particularly well suited for infection tracing, selection of strains for vaccine inclusion, and risk assessment. The ability of the assay to separate isolates into geographically linked and/or possibly host-specific clusters reflects its potential utility for maintenance of national biosecurity. The MLVA is internationally applicable and robust, and it provides clear, unambiguous, and easily interpreted results. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed from a harvested colony within a single working day. As the resulting MLVA profiles are readily portable, any strain may rapidly be placed in a global epizootiological context.
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http://dx.doi.org/10.1128/AEM.00730-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070765PMC
August 2018

Gibson Assembly facilitates bacterial allelic exchange mutagenesis.

J Microbiol Methods 2018 01 28;144:157-163. Epub 2017 Nov 28.

The University of Queensland, School of Biological Sciences and Centre for Marine Science, St Lucia Campus, Brisbane, Queensland 4072, Australia. Electronic address:

Allelic exchange mutagenesis that relies on RecA-mediated homologous recombination up- and downstream from the targeted gene is a generalizable method of site-specific bacterial gene knock-out and knock-in. However, generation of a mutagenic DNA construct (alternative allele flanked by regions surrounding the gene target) and subsequent mutant selection are laborious procedures. Here we demonstrate allelic exchange knock-out facilitated by Gibson Assembly in Streptococcus iniae. Gibson Assembly allows rapid construction of a large allelic exchange cassette simultaneous with cloning, as well as rapid reconstruction of complete recombinant vector sequence when required. Additionally, we show that during two-step mutant selection, absence of recombination at one of the homologous regions (single cross-over) might be rapidly detected by colony PCR of meroploid clones and resolved by extension/shifting of corresponding sequence in DNA construct. The combination of Gibson Assembly for mutagenic DNA construction/redesign with colony PCR screening of meroploids to detect recombination at both sides of the exchange target may significantly accelerate generation of chromosomal mutants in a wide range of bacterial taxa.
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http://dx.doi.org/10.1016/j.mimet.2017.11.023DOI Listing
January 2018

Whole genome analysis of isolated over 27 years in Australia and New Zealand reveals geographical endemism over multiple lineages and recent evolution under host selection.

Microb Genom 2016 11 30;2(11):e000095. Epub 2016 Nov 30.

4​Department of Primary Industries Parks Water & Environment (DPIPWE), Kings Meadows, Launceston, TAS 7249, Australia.

is a salmonid pathogen with widespread distribution in cool-temperate waters including Australia and New Zealand, two isolated environments with recently developed salmonid farming industries. Phylogenetic comparison of 58 isolates from Australia, New Zealand, USA, Chile, Finland and China based on non-recombinant core genome SNPs revealed multiple deep-branching lineages, with a most recent common ancestor estimated at 18 500 years BP (12 355-24 757 95% HPD) and evidence of Australasian endemism. Evolution within the Tasmanian Atlantic salmon serotype O1b lineage has been slow, with 63 SNPs describing the variance over 27 years. Isolates from the prevailing lineage are poorly/non-motile compared to a lineage pre-vaccination, introduced in 1997, which is highly motile but has not been isolated since from epizootics. A non-motile phenotype has arisen independently in Tasmania compared to Europe and USA through a frameshift in , encoding the ATPase of the flagella cluster. We report for the first time lipopolysaccharide O-antigen serotype O2 isolates in Tasmania. This phenotype results from deletion of the O-antigen cluster and consequent loss of high-molecular-weight O-antigen. This phenomenon has occurred independently on three occasions on three continents (Australasia, North America and Asia) as O2 isolates from the USA, China and Tasmania share the O-antigen deletion but occupy distant lineages. Despite the European and North American origins of the Australasian salmonid stocks, the lineages of in Australia and New Zealand are distinct from those of the northern hemisphere, suggesting they are pre-existing ancient strains that have emerged and evolved with the introduction of susceptible hosts following European colonization.
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http://dx.doi.org/10.1099/mgen.0.000095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320707PMC
November 2016

Immune transcriptome reveals the mincle C-type lectin receptor acts as a partial replacement for TLR4 in lipopolysaccharide-mediated inflammatory response in barramundi (Lates calcarifer).

Mol Immunol 2017 03 15;83:33-45. Epub 2017 Jan 15.

School of Biological Sciences, The University of Queensland, St. Lucia, QLD 4072, Australia. Electronic address:

Fish represent the most diverse and abundant extant vertebrate infraclass. They are also one of the earliest divergent phyla with adaptive immunity based on antigen recognition by MHC and immunoglobulin. The aquaculture industry, which currently provides more than half of the fish for human consumption globally, has successfully exploited the adaptive immune system of fish through mass vaccination programs. However, vaccination against highly diverse antigens, mostly carbohydrates, such as capsular polysaccharides and lipopolysaccharide (LPS) is challenging. Fish have a subdued innate response to LPS, but adaptive response is generally high and type-specific. To better understand the link between initial innate response and early onset of adaptive immunity to carbohydrate antigens in the perciform barramundi (Lates calcarifer), an immune transcriptome was prepared from pronephros and spleen following vaccination with LPS and peptidoglycan. From 163,661 transcripts derived by Illumina mRNA-Seq, most grouped in neuronal, endocrine or immune system categories, suggesting a close relationship between the three systems. Moreover, digestive enzyme transcripts in spleen appeared to be highly inducible in barramundi. Most of the known TLRs were transcribed in the barramundi spleen and HK transcriptome, with the notable exception of TLR4, which is primarily responsible for LPS recognition in mammals. Several C-type lectin receptors were also identified, including CD209, CD205, and CLEC4E (Mincle). As Mincle has been shown to bind LPS and is abundant on dendritic cells, its role in response to LPS in barramundi was further investigated. A high dose of LPS induced TNF-alpha expression via Mincle. However, IL-6 regulation, whilst still regulated in response to LPS, did not depend upon the Mincle pathway, suggesting other routes of activation. This study thus suggests that Mincle acts as a partial substitute for TLR4 in barramundi in the processing of LPS.
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http://dx.doi.org/10.1016/j.molimm.2017.01.010DOI Listing
March 2017

Transcriptome analysis of grey mullet (Mugil cephalus) after challenge with Lactococcus garvieae.

Fish Shellfish Immunol 2016 Nov 5;58:593-603. Epub 2016 Oct 5.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, 91201, Taiwan, ROC. Electronic address:

Grey mullet (Mugil cephalus) is an economically important fish species in Taiwan mariculture industry. Moreover, grey mullet are common hosts of a bacterial infection by Lactococcus garvieae. However, until now the information related to the immune system of grey mullet is unclear. Therefore, to understand the molecular basis underlying the host immune response to L. garvieae infection, Illumina HiSeq™ 2000 was used to analyse the head kidney and spleen transcriptome of infected grey mullet. De novo assembly of paired-end reads yielded 55,203 unigenes. Comparative analysis of the expression profiles between bacterial challenge fish and control fish identified a total of 7192 from head kidney and 7280 in spleen differentially expressed genes (P < 0.05), including 4211 upregulated genes and 2981 downregulated genes in head kidney, while in spleen 3598 genes were upregulated and 3682 downregulated. A significant enrichment analysis of these differentially expressed genes (DEG) in spleen and head kidney revealed major immune-related pathways, including complement and coagulation cascades, Toll-like receptor signalling, and antigen processing and presentation. Moreover, selected DEGs were validated using qPCR. Altogether, the results obtained on immune-related genes may allow for a better understanding of immunity in grey mullet to Lactococcus garvieae, carrying out detailed functional analysis of these genes and developing strategies for efficient immune protection against infections in grey mullet.
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http://dx.doi.org/10.1016/j.fsi.2016.10.006DOI Listing
November 2016

A reliable method for enrichment of neutrophils from peripheral blood in barramundi (Lates calcarifer).

Fish Shellfish Immunol 2016 Nov 15;58:174-176. Epub 2016 Sep 15.

School of Biological Sciences and Centre for Marine Science, The University of Queensland, St Lucia Campus, Brisbane, QLD, 4072, Australia.

Neutrophils are a short-lived, terminally differentiated, innate immune cell, that are critical first responders during infection. Research into neutrophil-pathogen interactions in fish has primarily employed cells derived from the pro-nephros and nephros. Since these sites are also the location of neutrophil and other immune cell development, there may be some ambiguity in maturation and functional ability of these cells, and difficulty in differentiating the effects of neutrophils from those of macrophages and monocytes. In contrast, peripheral blood circulating neutrophils are mature and ready to respond, thus it may be more physiologically relevant to use these cells for immune studies when evaluating interactions with blood-borne pathogens. The enrichment of tropical, euryhaline fish blood cells cannot follow classic mammalian enrichment methods for several reasons: Fish have nucleated red blood cells (RBC's), a high number of reticulocytes, a very low number of granulocytic leukocytes and an osmotic tolerance, rendering techniques such as water lysis ineffective. Enrichment of neutrophils, while minimizing RBC contamination, is imperative for studies where luminescence or fluorescence signals may be confounded by background from an overabundance of RBC's. We have optimized a method for enriching neutrophils from peripheral blood, with an initial settlement step employing 6% dextran (Mr 450,000-650,000), for 30-60 min at room temperature, followed by density separation on an 8-step Percoll density gradient. This method provides a cell suspension comprising 20-50% neutrophils, free of contamination from reticulocytes. These are then suitable for luminometric or fluorometric downstream analyses.
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http://dx.doi.org/10.1016/j.fsi.2016.09.028DOI Listing
November 2016

Streptococcus iniae cpsG alters capsular carbohydrate composition and is a cause of serotype switching in vaccinated fish.

Vet Microbiol 2016 Sep 17;193:116-24. Epub 2016 Aug 17.

The University of Queensland, School of Biological Sciences and Centre for Marine Science, Brisbane, Queensland, Australia. Electronic address:

Streptococcus iniae causes septicaemia and meningitis in marine and freshwater fish wherever they are farmed in warm-temperate and tropical regions. Although serotype specific, vaccination with bacterins (killed bacterial cultures) is largely successful and vaccine failure occurs only occasionally through emergence of new capsular serotypes. Previously we showed that mutations in vaccine escapes are restricted to a limited repertoire of genes within the 20-gene capsular polysaccharide (cps) operon. cpsG, a putative UDP-galactose 4-epimerase, has three sequence types based on the insertion or deletion of the three amino acids leucine, serine and lysine in the substrate binding site of the protein. To elucidate the role of cpsG in capsular polysaccharide (CPS) biosynthesis and capsular composition, we first prepared isogenic knockout and complemented mutants of cpsG by allelic exchange mutagenesis. Deletion of cpsG resulted in changes to colony morphology and cell buoyant density, and also significantly decreased galactose content relative to glucose in the capsular polysaccharide as determined by GC-MS, consistent with epimerase activity of CpsG. There was also a metabolic penalty of cpsG knockout revealed by slower growth in complex media, and reduced proliferation in whole fish blood. Moreover, whilst antibodies raised in fish against the wild type cross-reacted in whole cell and cps ELISA, they did not cross-opsonise the mutant in a peripheral blood neutrophil opsonisation assay, consistent with reported vaccine escape. We have shown here that mutation in cpsG results in altered CPS composition and this in turn results in poor cross-opsonisation that explains some of the historic vaccination failure on fish farms in Australia.
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http://dx.doi.org/10.1016/j.vetmic.2016.08.011DOI Listing
September 2016

Identification of Barramundi (Lates calcarifer) DC-SCRIPT, a Specific Molecular Marker for Dendritic Cells in Fish.

PLoS One 2015 14;10(7):e0132687. Epub 2015 Jul 14.

The University of Queensland, School of Biological Sciences and Centre for Marine Science, Brisbane, Queensland, 4072, Australia.

Antigen presentation is a critical step bridging innate immune recognition and specific immune memory. In mammals, the process is orchestrated by dendritic cells (DCs) in the lymphatic system, which initiate clonal proliferation of antigen-specific lymphocytes. However, fish lack a classical lymphatic system and there are currently no cellular markers for DCs in fish, thus antigen-presentation in fish is poorly understood. Recently, antigen-presenting cells similar in structure and function to mammalian DCs were identified in various fish, including rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio). The present study aimed to identify a potential molecular marker for DCs in fish and therefore targeted DC-SCRIPT, a well-conserved zinc finger protein that is preferentially expressed in all sub-types of human DCs. Putative dendritic cells were obtained in culture by maturation of spleen and pronephros-derived monocytes. DC-SCRIPT was identified in barramundi by homology using RACE PCR and genome walking. Specific expression of DC-SCRIPT was detected in barramundi cells by Stellaris mRNA FISH, in combination with MHCII expression when exposed to bacterial derived peptidoglycan, suggesting the presence of DCs in L. calcarifer. Moreover, morphological identification was achieved by light microscopy of cytospins prepared from these cultures. The cultured cells were morphologically similar to mammalian and trout DCs. Migration assays determined that these cells have the ability to move towards pathogens and pathogen associated molecular patterns, with a preference for peptidoglycans over lipopolysaccharides. The cells were also strongly phagocytic, engulfing bacteria and rapidly breaking them down. Barramundi DCs induced significant proliferation of responder populations of T-lymphocytes, supporting their role as antigen presenting cells. DC-SCRIPT expression in head kidney was higher 6 and 24 h following intraperitoneal challenge with peptidoglycan and lipopolysaccharide and declined after 3 days relative to PBS-injected controls. Relative expression was also lower in the spleen at 3 days post challenge but increased again at 7 days. As DC-SCRIPT is a constitutively expressed nuclear receptor, independent of immune activation, this may indicate initial migration of immature DCs from head kidney and spleen to the injection site, followed by return to the spleen for maturation and antigen presentation. DC-SCRIPT may be a valuable tool in the investigation of antigen presentation in fish and facilitate optimisation of vaccines and adjuvants for aquaculture.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132687PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501824PMC
May 2016

Resistance of Black-lip learl oyster, Pinctada margaritifera, to infection by Ostreid herpes virus 1μvar under experimental challenge may be mediated by humoral antiviral activity.

Fish Shellfish Immunol 2015 May 21;44(1):232-40. Epub 2015 Feb 21.

The University of Queensland, School of Biological Sciences and Centre for Marine Science, Brisbane, Queensland 4072, Australia.

Ostreid herpesvirus 1 (OsHV-1) has induced mass mortalities of the larvae and spat of Pacific oysters, Crassostrea gigas, in Europe and, more recently, in Oceania. The production of pearls from the Black-lip pearl oyster, Pinctada margaritifera, represents the second largest source of income to the economies of French Polynesia and many Pacific Island nations that could be severely compromised in the event of a disease outbreak. Coincidentally with the occurrence of OsHV-1 in the southern hemisphere, C. gigas imported from New Zealand and France into French Polynesia tested positive for OsHV-1. Although interspecies viral transmission has been demonstrated, the transmissibility of OsHV-1 to P. margaritifera is unknown. We investigated the susceptibility of juvenile P. margaritifera to OsHV-1 μvar that were injected with tissue homogenates sourced from either naturally infected or healthy C. gigas. The infection challenge lasted 14 days post-injection (dpi) with sampling at 0, 1, 2, 3, 5, 7 and 14 days. Mortality rate, viral prevalence, and cellular immune responses in experimental animals were determined. Tissues were screened by light microscopy and TEM. Pacific oysters were also challenged and used as a positive control to validate the efficiency of OsHV-1 μvar infection. Viral particles and features such as marginated chromatin and highly electron dense nuclei were observed in C. gigas but not in P. margaritifera. Mortality rates and hemocyte immune parameters, including phagocytosis and respiratory burst, were similar between challenged and control P. margaritifera. Herpesvirus-inhibiting activity was demonstrated in the acellular fraction of the hemolymph from P. margaritifera, suggesting that the humoral immunity is critical in the defence against herpesvirus in pearl oysters. Overall, these results suggest that under the conditions of the experimental challenge, P. margaritifera was not sensitive to OsHV-1 μvar and was not an effective host/carrier. The nature and spectrum of activity of the humoral antiviral activity is worthy of further investigation.
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http://dx.doi.org/10.1016/j.fsi.2015.02.026DOI Listing
May 2015

Transfer printing of self-folding polymer-metal bilayer particles.

ACS Appl Mater Interfaces 2014 Dec 4;6(24):22695-700. Epub 2014 Dec 4.

Department of Macromolecular Science and Engineering, and ‡Department of Electrical Engineering and Computer Science, Case Western Reserve University , Cleveland, Ohio 44106, United States.

A simple and robust alternative for fabricating stimuli-responsive 2D self-folding films was introduced. The approach combines metal-sputtering, layer-by-layer assembly of polyelectrolytes, and transfer-printing of the bilayer film onto a substrate coated with a sacrificial layer. With this technique, self-folding bilayer films can be fabricated without using harsh chemical etchants, complicated chemical synthesis, or complex lithographic techniques. Upon release, the microsized 2D film is shown to reconfigure into a 3D structure caused by a mismatch in the properties of the individual layers. The actuation of the bilayer film can be triggered by partial swelling due to absorption of water or by partial expansion of one of the layers due to an increase in temperature.
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http://dx.doi.org/10.1021/am5068172DOI Listing
December 2014

Characteristics of cyprinid herpesvirus 3 in different phases of infection: implications for disease transmission and control.

Virus Res 2014 Aug 3;188:45-53. Epub 2014 Apr 3.

CSIRO Animal, Food and Health Sciences, Australian Animal Health Laboratory, Geelong, VIC 3220, Australia.

Koi herpesvirus disease (KHVD) is an emerging and highly contagious viral disease of koi and common carp (Cyprinus carpio), causing mass mortalities and huge economic losses to the carp aquaculture industry. The disease has spread rapidly to 28 countries worldwide. However, mechanisms of koi herpesvirus (species Cyprinid herpesvirus 3; CyHV-3) transmission remain unclear. A potential experimental model of CyHV-3 infection in carp was used to characterise CyHV-3 in different phases of infection and to demonstrate that CyHV-3 persists in survivor fish and has the capacity to reactivate and transmit the disease to healthy fish. During acute infection, which occurred when fish were maintained at 22°C, viral genes were abundantly expressed and infectious virus was produced in association with tissue damage, clinical disease and mortality. In fish maintained at a lower temperature (11°C), viral DNA was present but viral gene expression was absent or greatly restricted, infectious virus was not recovered and there was no evidence of disease. Productive replication was re-initiated following an increase in water temperature to 22°C, resulting in 45% mortality. Shedding of reactivated virus killed 75% of cohabitating naïve fish, suggesting a potential risk for disease transmission.
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http://dx.doi.org/10.1016/j.virusres.2014.03.024DOI Listing
August 2014

Hemiuroid trematode sporocysts are undetected by hemocytes of their intermediate host, the ark cockle Anadara trapezia: potential role of surface carbohydrates in successful parasitism.

Fish Shellfish Immunol 2013 Dec 23;35(6):1937-47. Epub 2013 Oct 23.

The University of Queensland, School of Biological Sciences and Centre for Marine Science, Brisbane, QLD 4072, Australia.

In order to establish a successful relationship with their hosts, parasites must subvert or evade immune defences. Cockle Anadara trapezia and Sydney Rock oyster (SRO) Saccostrea glomerata live in the same location but only ark cockles are infected by sporocysts of hemiuroid trematode. This provides an opportunity to explore differing interactions between the parasite and the immune system of susceptible and refractive hosts. Rapid migration and encapsulation of sporocysts was observed by SRO hemocytes but not by cockle hemocytes. This migration/encapsulation was inhibited by N-acetylglucosamine or N-acetylgalactosamine but not by the other sugars, implicating specific surface carbohydrates in immune detection. Effector responses of hemocytes were investigated in vitro in terms of production of reactive oxygen production (ROS). Hemocytes of both species strongly reacted to Zymosan, but only SRO hemocytes responded to live sporocysts. Neither species' hemocytes produced ROS in the presence of dead/fixed sporocysts, and there was no suppression of Zymosan-induced respiratory burst by sporocysts. This suggests that immune escape is mediated by avoiding encapsulation, perhaps through molecular mimicry. Membrane-shaving with proteases indicated that sporocyst surface proteins are not a key factors in hemocytic detection. Surface carbohydrates of SRO and cockle hemocytes and of sporocysts were profiled with a panel of biotinylated lectins. This revealed substantial differences between cockle and SRO hemocytes, but greater similarity between cockle hemocytes and sporocysts. Results suggest that surface carbohydrates play an integral role in hemocyte immunorecognition and that surface carbohydrate molecular mimicry is a potential strategy for immune evasion in cockles by hemiuroid trematode sporocysts.
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http://dx.doi.org/10.1016/j.fsi.2013.09.040DOI Listing
December 2013

Effect of a hemiuroid trematode on the hemocyte immune parameters of the cockle Anadara trapezia.

Fish Shellfish Immunol 2013 Sep 16;35(3):951-6. Epub 2013 Jul 16.

University of Queensland, School of Biological Science and Centre for Marine Science, Brisbane, QLD 4072, Australia.

When a trematode parasite penetrates a potential molluscan host, it has to circumvent the host's internal defense system. In molluscs, the primary effector cells of this system are the hemocytes which orchestrate many of the cellular and humoral immune functions. Survival of the parasite can occur only in the absence of a successful immune response, and continued development only if the host is physiologically suitable. This study investigated hemocytic response against asexual stages of a hemiuroid trematode by its host, the marine bivalve Anadara trapezia. Hemocyte characteristic (type, morphology) and function (mortality, phagocytosis and oxidative activity) were analyzed by flow cytometry in parasitized and non-parasitized cockles. A. trapezia possesses two types of hemocytes: amebocytes and erythrocytes. Analysis of histological section showed that there was no host hemocytic response around hemiuroid sporocysts. The infection induced a significant increase of the total circulating hemocytes with a higher proportion of erythrocytes relative to amebocytes, coupled with a lower phagocytosis rate and a statistically non-significant decrease of the intracellular oxidative activity. No significant differences were observed in hemocyte size and complexity, mortality, or phagocytic capacity. Our results indicate that in A. trapezia, hemiuroids modulate the immune response by increasing the number of circulating hemocytes and decreasing phagocytosis.
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http://dx.doi.org/10.1016/j.fsi.2013.07.010DOI Listing
September 2013

Dietary soybean protein concentrate-induced intestinal disorder in marine farmed Atlantic salmon, Salmo salar is associated with alterations in gut microbiota.

Vet Microbiol 2013 Sep 10;166(1-2):286-92. Epub 2013 Jun 10.

The University of Queensland, School of Biological Sciences and Centre for Marine Science, Brisbane 4067, Australia.

The aquaculture industry has made substantial progress in reducing the fishmeal content of feeds for carnivorous species, driven by demand for improved sustainability and reduced cost. Soybean protein concentrate (SPC) is an attractive replacement for fishmeal, but intestinal disorders have been reported in Atlantic salmon (Salmo salar) fed these diets at high seawater temperatures, with preliminary evidence suggesting SPC induces these disorders by altering the intestinal microbiota. We compared the intestinal microbiota of marine-farmed S. salar fed experimental diets with varying levels of SPC in mid- and late-summer. Terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA clone library analysis revealed the microbiota adherent to the intestinal tract of salmon is complex at the population level, but simple and highly variable at the individual level. Temporal changes were observed with the bacterial diversity increasing in the intestinal tract in late summer. A Verrucomicrobia was the most frequently observed ribotype in early summer, whilst an Aliivibrio was the most frequently observed ribotype in late summer. Feeding SPC to salmon increased the bacterial diversity of the intestinal tract and resulted in the presence of bacteria not normally associated with marine fish (Escherichia and Propionibacterium). These diet-induced changes to the intestinal-microbiome could be ameliorated by inclusion of a prebiotic (mannan-oligosaccharide or MOS) to the diet. None of the experimental diets induced inflammation of the intestine as assessed by histopathology and expression of inflammatory cytokines. Our results support the "dysbiosis" hypothesis that SPC adversely affects the intestinal microbiota of Atlantic salmon.
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http://dx.doi.org/10.1016/j.vetmic.2013.05.009DOI Listing
September 2013

Parasites of QX-resistant and wild-type Sydney rock oysters (Saccostrea glomerata) in Moreton Bay, SE Queensland, Australia: diversity and host response.

J Invertebr Pathol 2013 Mar 27;112(3):273-7. Epub 2012 Dec 27.

The University of Queensland, School of Biological Science and Centre for Marine Science, Brisbane, QLD 4072, Australia.

Wild caught (WC) and QX resistant (QXR) Sydney rock oysters were introduced at North Stradbroke Island and Pimpama River, SE Queensland, Australia, and sampled monthly during 1 year. Three groups of parasites/diseases were identified by observation of histological sections: (1) Marteilia sydneyi (Queensland unknown (QX) disease) and Steinhausia sp. (Microsporidia) characterized by a high prevalence and deleterious impact on the host; (2) disseminated neoplasia and the trematode Proctoeces sp. characterized by low prevalence but deleterious effects on the host; (3) parasites or symbionts with no detectable effect on the host: trematodes, ciliates, turbellarians and metacestodes. Mortality rates were similar between both oyster lines but higher at Pimpama River (reaching around 90%) than Stradbroke Island, mostly because of QX disease and, to a lesser extent, to the unfavourable environmental conditions of the summer 2010-2011. Lower prevalences of QX disease at Stradbroke Island probably related to the relative lack of intermediate hosts of the parasite and to lower freshwater input. Surprisingly, no difference in prevalence of QX disease was observed between the two oyster lines.
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http://dx.doi.org/10.1016/j.jip.2012.12.006DOI Listing
March 2013

Evolution of the capsular operon of Streptococcus iniae in response to vaccination.

Appl Environ Microbiol 2012 Dec 21;78(23):8219-26. Epub 2012 Sep 21.

School of Biological Sciences and Centre for Marine Science, The University of Queensland, Brisbane, Australia.

Streptococcus iniae causes severe septicemia and meningitis in farmed fish and is also occasionally zoonotic. Vaccination against S. iniae is problematic, with frequent breakdown of protection in vaccinated fish. The major protective antigens in S. iniae are the polysaccharides of the capsule, which are essential for virulence. Capsular biosynthesis is driven and regulated by a 21-kb operon comprising up to 20 genes. In a long-term study, we have sequenced the capsular operon of strains that have been used in autogenous vaccines across Australia and compared it with the capsular operon sequences of strains subsequently isolated from infected vaccinated fish. Intriguingly, strains isolated from vaccinated fish that subsequently become infected have coding mutations that are confined to a limited number of genes in the cps operon, with the remainder of the genes in the operon remaining stable. Mutations in strains in diseased vaccinated fish occur in key genes in the capsular operon that are associated with polysaccharide configuration (cpsG) and with regulation of biosynthesis (cpsD and cpsE). This, along with high ratios of nonsynonymous to synonymous mutations within the cps genes, suggests that immune response directed predominantly against capsular polysaccharide may be driving evolution in a very specific set of genes in the operon. From these data, it may be possible to design a simple polyvalent vaccine with a greater operational life span than the current monovalent killed bacterins.
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http://dx.doi.org/10.1128/AEM.02216-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497394PMC
December 2012

The conserved surface M-protein SiMA of Streptococcus iniae is not effective as a cross-protective vaccine against differing capsular serotypes in farmed fish.

Vet Microbiol 2013 Feb 1;162(1):151-9. Epub 2012 Sep 1.

The University of Queensland, School of Biological Sciences and Centre for Marine Science, Brisbane, QLD 4072, Australia.

Streptococcus iniae causes invasive infections in fresh and saltwater fish and occasional zoonoses. Vaccination against S. iniae is complicated by serotypic variation determined by capsular polysaccharide. A potential target for serologically cross-protective vaccines is the M-like protein SiMA, an essential virulence factor in S. iniae that is highly conserved amongst virulent strains. The present study determined how SiMA is regulated and investigated potential as a cross-protective vaccine for fish. Electrophoretic mobility shift suggested that SiMA is regulated by the multigene regulator Mgx via a binding site in the -35 region of the simA promoter. Moreover, expression of simA and mgx was highly correlated, with the highest level of simA and mgx expression during exponential growth under iron limitation (20-fold increase in relative expression compared to growth in Todd-Hewitt broth). Based on these results, a vaccination and challenge experiment was conducted in barramundi (Lates calcarifer) to determine whether SiMA is protective against S. iniae infection and cross-protective against a different capsular serotype. The challenge resulted in 60% mortality in control fish. Formalin-killed bacterins prepared from the challenge strain resulted in 100% protection, whereas bacterins prepared from a serotypically heterologous strain resulted in significantly reduced protection, even when culture conditions were manipulated to optimise SiMA expression. Moreover, recombinant SiMA protein was not protective against the challenge strain in spite of eliciting specific antibody response in vaccinated fish. Specific antibody did not increase oxidative activity or phagocytosis by barramundi macrophages. Indeed incubating S. iniae with antisera significantly reduced phagocytosis. Lack of specific-antibody mediated opsonisation in spite of 100% protection against challenge with the homologous vaccine suggests that other immune parameters result in protection of challenged fish.
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http://dx.doi.org/10.1016/j.vetmic.2012.08.018DOI Listing
February 2013

Genetic analysis of Black Tiger shrimp (Penaeus monodon) across its natural distribution range reveals more recent colonization of Fiji and other South Pacific islands.

Ecol Evol 2012 Aug 22;2(8):2057-71. Epub 2012 Jul 22.

The Black Tiger shrimp (Penaeus monodon) has a natural distribution range from East Africa to the South Pacific Islands. Although previous studies of Indo-Pacific P. monodon have found populations from the Indian Ocean and Australasia to differ genetically, their relatedness to South Pacific shrimp remains unknown. To address this, polymorphisms at eight shared microsatellite loci and haplotypes in a 418-bp mtDNA-CR (control region) sequence were examined across 682 P. monodon from locations spread widely across its natural range, including the South Pacific islands of Fiji, Palau, and Papua New Guinea (PNG). Observed microsatellite heterozygosities of 0.82-0.91, allele richness of 6.85-9.69, and significant mtDNA-CR haplotype variation indicated high levels of genetic diversity among the South Pacific shrimp. Analysis of microsatellite genotypes using a Bayesian STRUCTURE method segregated Indo-Pacific P. monodon into eight distinct clades, with Palau and PNG shrimp clustering among others from Southeast Asia and eastern Australia, respectively, and Fiji shrimp clustering as a distinct group. Phylogenetic analyses of mtDNA-CR haplotypes delineated shrimp into three groupings, with shrimp from Fiji again being distinct by sharing no haplotypes with other populations. Depending on regional location, the genetic structures and substructures identified from the genotyping and mtDNA-CR haplotype phylogeny could be explained by Metapopulation and/or Member-Vagrant type evolutionary processes. Neutrality tests of mutation-drift equilibrium and estimation of the time since population expansion supported a hypothesis that South Pacific P. monodon were colonized from Southeast Asia and eastern Australia during the Pleistocene period over 60,000 years ago when land bridges were more expansive and linked these regions more closely.
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http://dx.doi.org/10.1002/ece3.316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434007PMC
August 2012

Koi herpesvirus encodes and expresses a functional interleukin-10.

J Virol 2012 Nov 15;86(21):11512-20. Epub 2012 Aug 15.

CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Victoria, Australia.

Koi herpesvirus (KHV) (species Cyprinid herpesvirus 3) ORF134 was shown to transcribe a spliced transcript encoding a 179-amino-acid (aa) interleukin-10 (IL-10) homolog (khvIL-10) in koi fin (KF-1) cells. Pairwise sequence alignment indicated that the expressed product shares 25% identity with carp IL-10, 22 to 24% identity with mammalian (including primate) IL-10s, and 19.1% identity with European eel herpesvirus IL-10 (ahvIL-10). In phylogenetic analyses, khvIL-10 fell in a divergent position from all host IL-10 sequences, indicating extensive structural divergence following capture from the host. In KHV-infected fish, khvIL-10 transcripts were observed to be highly expressed during the acute and reactivation phases but to be expressed at very low levels during low-temperature-induced persistence. Similarly, KHV early (helicase [Hel] and DNA polymerase [DNAP]) and late (intercapsomeric triplex protein [ITP] and major capsid protein [MCP]) genes were also expressed at high levels during the acute and reactivation phases, but only low-level expression of the ITP gene was detected during the persistent phase. Injection of khvIL-10 mRNA into zebrafish (Danio rerio) embryos increased the number of lysozyme-positive cells to a similar degree as zebrafish IL-10. Downregulation of the IL-10 receptor long chain (IL-10R1) using a specific morpholino abrogated the response to both khvIL-10 and zebrafish IL-10 transcripts, indicating that, despite the structural divergence, khvIL-10 functions via this receptor. This is the first report describing the characteristics of a functional viral IL-10 gene in the Alloherpesviridae.
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http://dx.doi.org/10.1128/JVI.00957-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486318PMC
November 2012

Gender differences in hemocyte immune parameters of bivalves: the Sydney rock oyster Saccostrea glomerata and the pearl oyster Pinctada fucata.

Fish Shellfish Immunol 2012 Jul 25;33(1):138-42. Epub 2012 Apr 25.

School of Biological Sciences, The University of Queensland, Brisbane, QLD 4072, Australia.

Many authors have highlighted a high inter-individual variability in immune parameters of marine bivalves. A high number of studies have reported the impact of external factors on hemocytes immune parameters such as temperature, salinity, pollutants or pathogens. However, only a few of them considered the impact of intrinsic parameters such as sex. Therefore, the present study assessed the impact of gender on hemocytes functions on two marine bivalves. Our results led to the conclusion that the gender contributes to this inter-individual variability. When studying the impact of an environmental variable, a pathogen or a pollutant, the sex of each animal should be determined and taken into account in the analysis and interpretation of immune parameters.
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http://dx.doi.org/10.1016/j.fsi.2012.04.007DOI Listing
July 2012