Transforming the untransformable with knockout minicircles: High-efficiency transformation and vector-free allelic exchange knockout in the fish pathogen Photobacterium damselae.

Gene inactivation studies are critical in pathogenic bacteria, where insights into species biology can guide the development of vaccines and treatments. Allelic exchange via homologous recombination is a generic method of targeted gene editing in bacteria. However, generally applicable protocols are lacking, and suboptimal approaches are often used for nonstandard but epidemiologically important species.

Biolayer interferometry is insufficiently sensitive for direct measurement of IgM quantity and avidity in Atlantic salmon (Salmo salar) serum.

Quantification of specific antibodies underpins the assessment of adaptive immunity in response to vaccination or infection and is performed by enzyme-linked immunosorbent assay (ELISA). A biolayer interferometry (BLI) assay was recently developed that simultaneously quantifies IgM antibodies and their avidity in giant grouper (Epinephelus lanceolatus) sera and proved to be a robust, repeatable and more high-throughput alternative to ELISA [1]. Here we attempted to optimise a similar single-step BLI assay using an Octet HTX instrument to quantify IgM specific to the hapten 2,4-dinitrophenol (DNP) in serum from Atlantic salmon (Salmo salar) primed and boosted with DNP conjugated to keyhole limpet hemocyanin.

The Evolution of a Specialized, Highly Virulent Fish Pathogen through Gene Loss and Acquisition of Host-Specific Survival Mechanisms.

Photobacterium damselae comprises two subspecies, P. damselae subsp. damselae and P.

Creating value from purple phototrophic bacteria via single-cell protein production.

Mixed culture purple phototrophic bacteria (PPB) is a rapidly emerging technology for resource recovery from wastewaters. PPB biomass can be used as single-cell protein, with a high protein content complemented by value-added components (e.g.

A high-quality reference genome for the fish pathogen .

Fish mortality caused by is a major economic problem in aquaculture in warm and temperate regions globally. There is also risk of zoonotic infection by through handling of contaminated fish. In this study, we present the complete genome sequence of strain QMA0248, isolated from farmed barramundi in South Australia.

Molecular Investigation of Recurrent Epizootics Affecting Coral Reef Fish on an Oceanic Island Suggests at Least Two Distinct Emergence Events.

is an emerging zoonotic pathogen of increasing concern for aquaculture and has caused several epizootics in reef fishes from the Caribbean, the Red Sea and the Indian Ocean. To study the population structure, introduction pathways and evolution of over recurring epizootics on Reunion Island, we developed and validated a Multi Locus Sequence Typing (MLST) panel using genomic data obtained from 89 isolates sampled during epizootics occurring over the past 40years in Australia, Asia, the United States, Israel and Reunion Island. We selected eight housekeeping loci, which resulted in the greatest variation across the main phylogenetic clades highlighted by the whole genomic dataset.

A single-step, high throughput, and highly reproducible method for measuring IgM quantity and avidity directly from fish serum via biolayer interferometry (BLI).

Quantification of specific antibody responses is critical in determining activation of MHCII-dependent immune memory and is generally performed by enzyme-linked immunosorbent assay (ELISA). Antibody avidity for a particular antigen is also informative of the quality of the adaptive immune response following vaccination. Avidity can be determined by chaotropic elution ELISA, pre-absorption ELISA, or surface plasmon resonance (SPR), although multimeric antibodies such as IgM are problematic for SPR.

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

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.

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.

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.

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).

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.

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

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.

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

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.

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

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.

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

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).

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

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.

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

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.

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

(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.

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

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.

Gibson Assembly facilitates bacterial allelic exchange mutagenesis.

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.

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).

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.

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

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.

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

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.

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

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.