Stimulation of MCM helicase activity by a Cdc6 protein in the archaeon Thermoplasma acidophilum

Replicative DNA helicases are ring-shaped hexamers that play an essential role in chromosomal DNA replication. They unwind the two strands of the duplex DNA and provide the single-stranded (ss) DNA substrate for the polymerase. The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in eukarya and archaea. The proteins of only a few archaeal organisms have been studied and revealed that although all have similar amino acid sequences and overall structures they differ in their biochemical properties. In this report the biochemical properties of the MCM protein from the archaeon Thermoplasma acidophilum is described. The enzyme has weak helicase activity on a substrate containing only a 3′-ssDNA overhang region and the protein requires a forked DNA structure for efficient helicase activity. It was also found that the helicase activity is stimulated by one of the two T.acidophilum Cdc6 homologues. This is an interesting observation as it is in sharp contrast to observations made with MCM and Cdc6 homologues from other archaea in which the helicase activity is inhibited when bound to Cdc6.publishedVersio

Comparing Body Density of Lumpfish (Cyclopterus lumpus) to Different Operational Welfare Indicators

Farmed lumpfish (Cyclopterus lumpus) are commonly used as cleaner fish in the salmonid aquaculture industry, but a knowledge gap exists with regards to their body density. Filling this knowledge gap is of importance, as the lumpfish has no swim bladder and thus relies on alternative methods for buoyancy, i.e., the body density difference between the fish and its surroundings. The aims of this study were to measure the body density of lumpfish and investigate the correlation between body density and different operational welfare indicators. A total of 138 lumpfish were sampled at five different aquaculture sites situated in the Faroe Islands. Weight in water and air was measured, body density was calculated, and operational welfare was assessed. The average body density of the juvenile lumpfish was 1.030 g mL−1. Fulton’s K, stomach score, and length were negatively correlated to body density, while the hepatosomatic index was positively correlated to body density. Liver colour was correlated to body density, but the groupings were too broad for a final definitive conclusion. The knowledge gained from this study might help the industry improve their understanding of the operational welfare indicators used for lumpfish. Additionally, the knowledge might also help the aquaculture industry improve their husbandry and feeding practices.publishedVersio

Neutrophils in Atlantic salmon (Salmo salar L.) are MHC class II+ and secret IL-12p40 upon bacterial exposure

Antigen-presentation via major histocompatibility complex (MHC) to T cells is the key event to initiate adaptive immune responses. In teleosts, as in mammals, the main types of professional antigen-presenting cells (APCs) are dendritic cells (DCs), monocytes/macrophages, and B cells. In the current study, flow cytometry, immunostaining and qPCR have been used to show that neutrophils in the teleost fish Atlantic salmon (Salmo salar L.) have antigen-presenting properties. The neutrophils were positive for MHC class II, CD83 and CD80/86, and upon in vitro bacterial exposure, gene expression analysis of purified neutrophils showed that IL-12p40, which is essential for proliferation of naïve T cells, was highly upregulated at both 6 and 24 h post bacterial exposure. Based on presence of MHC class II and upregulation of molecules involved in antigen presentation and T cell activation, we suggest that neutrophils in Atlantic salmon have potential to function as professional APCs. This work makes an important basis for further exploring the potential of using neutrophils to develop new, targeted immunoprophylactic measures.publishedVersio

Transcriptome-wide analyses of early immune responses in lumpfish leukocytes upon stimulation with poly(I:C)

Background: Both bacterial and viral diseases are a major threat to farmed fish. As the antiviral immune mechanisms in lumpfish (Cyclopterus lumpus L.) are poorly understood, lumpfish leukocytes were stimulated with poly(I:C), a synthetic analog of double stranded RNA, which mimic viral infections, and RNA sequencing was performed. Methods: To address this gap, we stimulated lumpfish leukocytes with poly(I:C) for 6 and 24 hours and did RNA sequencing with three parallels per timepoint. Genome guided mapping was performed to define differentially expressed genes (DEGs). Results: Immune genes were identified, and transcriptome-wide analyses of early immune responses showed that 376 and 2372 transcripts were significantly differentially expressed 6 and 24 hours post exposure (hpe) to poly(I:C), respectively. The most enriched GO terms when time had been accounted for, were immune system processes (GO:0002376) and immune response (GO:0006955). Analysis of DEGs showed that among the most highly upregulated genes were TLRs and genes belonging to the RIG-I signaling pathway, including LGP2, STING and MX, as well as IRF3 and IL12A. RIG-I was not identified, but in silico analyses showed that genes encoding proteins involved in pathogen recognition, cell signaling, and cytokines of the TLR and RIG-I signaling pathway are mostly conserved in lumpfish when compared to mammals and other teleost species. Conclusions: Our analyses unravel the innate immune pathways playing a major role in antiviral defense in lumpfish. The information gathered can be used in comparative studies and lay the groundwork for future functional analyses of immune and pathogenicity mechanisms. Such knowledge is also necessary for the development of immunoprophylactic measures for lumpfish, which is extensively cultivated for use as cleaner fish in the aquaculture for removal of sea lice from Atlantic salmon (Salmo salar L.).publishedVersio

Pharmacokinetics of florfenicol in lumpfish (Cyclopterus lumpus L.) after a single oral administration

Farming of lumpfish for biological removal of sea lice from farmed Atlantic salmon has expanded rapidly in Europe and Canada over the last 5–6 years and the lumpfish has become an economically important species. There are, however, health challenges associated with bacterial diseases. In recent years, there has been an increase in antibacterial treatments prescribed for this fish species despite a lack of knowledge regarding pharmacokinetics and effect of treatment with different antibiotics. The present study examined the uptake, tissue distribution, metabolism and elimination of the antibacterial agent florfenicol in lumpfish (Cyclopterus lumpus L.) following a single oral administration of 10 mg/kg fish given in feed. Plasma, head kidney, liver and muscle from six fish were sampled at each time point and analysed by liquid chromatography/mass spectrometry (LC-MS). Absorption was moderate for this drug characterised by a calculated peak plasma concentration (Cmax) of 3.55 μg/ml obtained after 21.2 hours (Tmax) and the elimination halflife (t1/2β) relatively extended in plasma at 30 hours. Area under curve (AUC) and AUC from 0 to 24 hours (AUC0-24h) were calculated to be 248 and 61 h μg/ml, respectively. Cmax was calculated to 2.99 μg/g in muscle, 2.54 μg/g in liver and 4.70 μg/g in head kidney with corresponding Tmax of 22.1, 26.4 and 19.4 h, respectively. The main metabolite, florfenicol-amine was found in low concentrations in plasma and all tissues examined. The minimum inhibition concentrations (MIC) for florfenicol of 28 of Aeromonas salmonicida isolates from diseased lumpfish ranged from 0.39 to 1.56 μg/ml. The pharmacokinetical data presented here make an important basis for efficient antibacterial treatment for lumpfish using florfenicol and for calculation of suitable withdrawal time. Knowledge of florfenicol pharmacokinetics, combined with determination of antibiotic resistance among fish pathogenic bacteria and the effect of antibacterial agents on diseased lumpfish in vivo are important for the welfare of lumpfish and prevention of resistant bacteria.publishedVersio

Pharmacokinetic Data Show That Oxolinic Acid and Flumequine Are Absorbed and Excreted Rapidly From Plasma and Tissues of Lumpfish

This study examined the uptake, tissue distribution and elimination of the antibacterial agents oxolinic acid and flumequine in lumpfish (Cyclopterus lumpus L.) by use of LC-MS/MS following a single oral administration of 25 mg/kg fish given in feed. Lumpfish are increasingly used as cleaner fish for removal of sea lice on commercially farmed salmon. The production of lumpfish is successful, but there are challenges with bacterial infections and the number of antibacterial treatments has increased in recent years. As the lumpfish is a novel species to farming, there is a need for pharmacokinetic data and establishment of protocols for efficient antibacterial treatment. The current study describes the pharmacokinetic properties of oxolinic acid and flumequine in lumpfish. Absorption of oxolinic acid was moderate and was characterized by a calculated peak plasma concentration (Cmax) of 2.12 μg/ml after 10.3 h (Tmax) and an elimination half-life (t1/2β) of 21 h. Area under curve (AUC) and AUC from 0 to 24 h (AUC0−24h) were calculated to be 60.9 and 34.0 h μg/ml, respectively. For flumequine, plasma Cmax was found to be 2.77 μg/ml after 7.7 h (Tmax) with t1/2β of 22 h. The area under the curve (AUC) and AUC from 0 to 24 h (AUC0−24) were calculated as 104.3 and 50.3 h μg/ml, respectively. Corresponding Cmax values in muscle, liver, and head-kidney for oxolinic acid were 4.01, 3.04, and, 4.68 μg/g, respectively and Tmax of 11.1, 9.2, and 10.0 h, respectively. For flumequine, Cmax values of 4.16, 4.01, and 7.48 μg/g were obtained in muscle, liver, and head kidney, respectively, with corresponding Tmax values of 10.2, 10.3, and 6.0 h. Antimicrobial susceptibility values as determined by minimum inhibitory concentration (MIC) analyses against 28 isolates of Aeromonas salmonicida isolated from diseased lumpfish ranged from 0.06 to 15 μg/ml for oxolinic acid and 0.024 to 6.25 μg/ml for flumequine. Bimodal distributions in susceptibility to both oxolinic acid and flumequine were observed. The combination of pharmacokinetic properties and MIC data make possible calculation of efficient treatment doses, which are needed to improve the welfare of lumpfish and minimize development of antibiotic resistant bacteria.publishedVersio

Genomic analysis of pasteurella atlantica provides insight on its virulence factors and phylogeny and highlights the potential of reverse vaccinology in aquaculture

Pasteurellosis in farmed lumpsuckers, Cyclopterus lumpus, has emerged as a serious disease in Norwegian aquaculture in recent years. Genomic characterization of the causative agent is essential in understanding the biology of the bacteria involved and in devising an efficient preventive strategy. The genomes of two clinical Pasteurella atlantica isolates were sequenced (≈2.3 Mbp), and phylogenetic analysis confirmed their position as a novel species within the Pasteurellaceae. In silico analyses revealed 11 genomic islands and 5 prophages, highlighting the potential of mobile elements as driving forces in the evolution of this species. The previously documented pathogenicity of P. atlantica is strongly supported by the current study, and 17 target genes were recognized as putative primary drivers of pathogenicity. The expression level of a predicted vaccine target, an uncharacterized adhesin protein, was significantly increased in both broth culture and following the exposure of P. atlantica to lumpsucker head kidney leucocytes. Based on in silico and functional analyses, the strongest gene target candidates will be prioritized in future vaccine development efforts to prevent future pasteurellosis outbreaks.publishedVersio

Stimulation of MCM helicase activity by a Cdc6 protein in the archaeon Thermoplasma acidophilum

Replicative DNA helicases are ring-shaped hexamers that play an essential role in chromosomal DNA replication. They unwind the two strands of the duplex DNA and provide the single-stranded (ss) DNA substrate for the polymerase. The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in eukarya and archaea. The proteins of only a few archaeal organisms have been studied and revealed that although all have similar amino acid sequences and overall structures they differ in their biochemical properties. In this report the biochemical properties of the MCM protein from the archaeon Thermoplasma acidophilum is described. The enzyme has weak helicase activity on a substrate containing only a 3′-ssDNA overhang region and the protein requires a forked DNA structure for efficient helicase activity. It was also found that the helicase activity is stimulated by one of the two T.acidophilum Cdc6 homologues. This is an interesting observation as it is in sharp contrast to observations made with MCM and Cdc6 homologues from other archaea in which the helicase activity is inhibited when bound to Cdc6

Thermoplasma acidophilum Cdc6 protein stimulates MCM helicase activity by regulating its ATPase activity

The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. In most archaeal species studied, the interaction between MCM and the initiator protein, Cdc6, inhibits helicase activity. To date, the only exception is the helicase and Cdc6 proteins from the archaeon Thermoplasma acidophilum. It was previously shown that when the Cdc6 protein interacts with MCM it substantially stimulates helicase activity. It is shown here that the mechanism by which the Cdc6 protein stimulates helicase activity is by stimulating the ATPase activity of MCM. Also, through the use of site-specific substitutions, and truncated and chimeric proteins, it was shown that an intact Cdc6 protein is required for this stimulation. ATP binding and hydrolysis by the Cdc6 protein is not needed for the stimulation. The data suggest that binding of Cdc6 protein to MCM protein changes the structure of the helicase, enhancing the catalytic hydrolysis of ATP and helicase activity