Proteomic and toxicological analysis of the response of dinoflagellate Alexandrium catenella to changes in NaNO3 concentration.

Dinoflagellates of the genus Alexandrium cause Harmful Algal Blooms (HABs) in coastal waters worldwide, damaging marine environments, aquaculture, and human health. They synthesize potent neurotoxic alkaloids known as PSTs (i.e., Paralytic Shellfish Toxins), the etiological agents of PSP (i.e., Paralytic Shellfish Poisoning). In recent decades, the eutrophication of coastal waters with inorganic nitrogen (e.g., nitrate, nitrite, and ammonia) has increased the frequency and scale of HABs. PSTs concentrations within Alexandrium cells can increase by up to 76% after a nitrogen enrichment event; however, the mechanisms that underlie their biosynthesis in dinoflagellates remains unclear. This study combines mass spectrometry, bioinformatics, and toxicology and investigates the expression profiles of PSTs in Alexandrium catenella grown in 0.4, 0.9 and 1.3 mM NaNO3. Pathway analysis of protein expression revealed that tRNA amino acylation, glycolysis, TCA cycle and pigment biosynthesis were upregulated in 0.4 mM and downregulated in 1.3 mM NaNO3 compared to those grown in 0.9 mM NaNO3. Conversely, ATP synthesis, photosynthesis and arginine biosynthesis were downregulated in 0.4 mM and upregulated in 1.3 mM NaNO3. Additionally, the expression of proteins involved in PST biosynthesis (sxtA, sxtG, sxtV, sxtW and sxtZ) and overall PST production like STX, NEO, C1, C2, GTX1-6 and dcGTX2 was higher at lower nitrate concentrations. Therefore, increased nitrogen concentrations increase protein synthesis, photosynthesis, and energy metabolism and decrease enzyme expression in PST biosynthesis and production. This research provides new clues about how the changes in the nitrate concentration can modulate different metabolic pathways and the expression of PST biosynthesis in toxigenic dinoflagellates.

Sodium Tungstate (NaW) Decreases Reactive Oxygen Species (ROS) Production in Cells: New Cellular Antioxidant.

Diabetic nephropathy (DN) is the leading cause of end-stage renal failure worldwide. Hyperglycemia generates reactive oxygen species (ROS), contributing to diabetic complications, especially in DN. Sodium Tungstate (NaW) is an effective antidiabetic agent for short and long-term treatments of both type 1 and type 2 diabetes models. In this study, we evaluated the effect of NaW on ROS production in bovine neutrophils incubated with platelet-activating factor (PAF) and in HK-2 cells induced by high glucose or H2O2. In addition, we evaluated the effect on iNOS expression in the type 1 diabetic rat model induced with streptozotocin (STZ). NaW inhibited ROS production in PAF-induced bovine neutrophils, and human tubular cells (HK-2) were incubated in high glucose or H2O2. In addition, NaW inhibited iNOS expression in glomeruli and tubular cells in the type 1 diabetic rat. This study demonstrates a new role for NaW as an active antioxidant and its potential use in treating DN.

Genomic and proteomic aspects of p57 protein from Renibacterium salmoninarum: Characteristics in virulence patterns.

Renibacterium salmoninarum is one of the oldest known fish bacterial pathogens. This Gram-positive bacterium is the causative agent of Bacterial Kidney Disease (BKD), a chronic infection that primarily infects salmonids at low temperatures. Externally, infected fish may show exophthalmos, skin blisters, ulcerations, and hemorrhages at the base of the fins and along the lateral line. Internally, the kidney, heart, spleen, and liver may show signs of inflammation. The best characterized virulence factor of R. salmoninarum is p57, a 57 kDa protein located on the bacterial cell surface and secreted into surrounding fish tissue. The p57 protein in fish is the main mediator in suppressing the immune system, reducing antibody production, and intervening in cytokine activity. In this review, we will discuss aspects such as single nucleotide polymorphisms (SNPs) that modify the DNA sequence, variants in the number of copies of MSA genes, physical-chemical properties of the signal peptides, and the limited iron conditions that can modify p57 expression and increase the virulence of R. salmoninarum.

Microencapsulation of Piscirickettsia salmonis Antigens for Fish Oral Immunization: Optimization and Stability Studies.

The development of fish oral vaccines is of great interest to the aquaculture industry due to the possibility of rapid vaccination of a large number of animals at reduced cost. In a previous study, we evaluated the effect of alginate-encapsulated Piscirickettsia salmonis antigens (AEPSA) incorporated in feed, effectively enhancing the immune response in Atlantic salmon (Salmo salar). In this study, we seek to characterize AEPSA produced by ionic gelation using an aerodynamically assisted jetting (AAJ) system, to optimize microencapsulation efficiency (EE%), to assess microparticle stability against environmental (pH, salinity and temperature) and gastrointestinal conditions, and to evaluate microparticle incorporation in fish feed pellets through micro-CT-scanning. The AAJ system was effective in obtaining small microparticles (d < 20 µm) with a high EE% (97.92%). Environmental conditions (pH, salinity and temperature) generated instability in the microparticles, triggering protein release. 62.42% of the protein content was delivered at the intestinal level after in vitro digestion. Finally, micro-CT-scanning images confirmed microparticle incorporation in fish feed pellets. In conclusion, the AAJ system is effective at encapsulating P. salmonis antigens in alginate with a high EE% and a size small enough to be incorporated in fish feed and produce an oral vaccine.

Effect of low-dose Piscirickettsia salmonis infection on haematological-biochemical blood parameters in Atlantic salmon (Salmo salar).

Piscirickettsia salmonis is the etiological agent of Piscirickettsiosis, a severe disease that affects Atlantic salmon (Salmo salar) farmed in Chile and many other areas (Norway, Scotland, Ireland, Canada and the USA). This study investigated the effects of low-dose P. salmonis infection (1 × 102 CFU/ml) on Atlantic salmon. In this study, we challenged fish with an isolated representative of the EM-90 genogroup via intraperitoneal injection for 42 days. Infected fish displayed decreased haematocrit and haemoglobin levels at day 13 post-infection, indicating erythropenia, haemolysis and haemodilution. Conversely, their white blood cell counts increased on days 13 and 21 post-infection. Additionally, their iron levels decreased from day 2 post-infection, indicating iron deficiency and an inability to retrieve stored iron before infection. Their magnesium levels also decreased at day 28 post-infection, possibly due to osmoregulatory problems. Also, we observed an increase in lactate dehydrogenase activity on days 5, 21, and 28 post-infection, suggesting early symptoms of hepatotoxicity. Later analyses determined a decrease in plasma glucose levels from day 2 post-infection. This may be attributed to the hypoxic conditions caused by P. salmonis, leading to an excess utilization of stored carbohydrates. Our results suggest that the blood parameters we studied are useful for monitoring the physiological status of Atlantic salmon infected with P. salmonis.

Protein-Based Vaccine Protect Against Piscirickettsia salmonis in Atlantic Salmon (Salmo salar).

An effective and economical vaccine against the Piscirickettsia salmonis pathogen is needed for sustainable salmon farming and to reduce disease-related economic losses. Consequently, the aquaculture industry urgently needs to investigate efficient prophylactic measures. Three protein-based vaccine prototypes against Piscirickettsia salmonis were prepared from a highly pathogenic Chilean isolate. Only one vaccine effectively protected Atlantic salmon (Salmo salar), in correlation with the induction of Piscirickettsia-specific IgM antibodies and a high induction of transcripts encoding pro-inflammatory cytokines (i.e., Il-1ß and TNF-α). In addition, we studied the proteome fraction protein of P. salmonis strain Austral-005 using multidimensional protein identification technology. The analyzes identified 87 proteins of different subcellular origins, such as the cytoplasmic and membrane compartment, where many of them have virulence functions. The other two prototypes activated only the innate immune responses, but did not protect Salmo salar against P. salmonis. These results suggest that the knowledge of the formulation of vaccines based on P. salmonis proteins is useful as an effective therapy, this demonstrates the importance of the different research tools to improve the study of the different immune responses, resistance to diseases in the Atlantic salmon. We suggest that this vaccine can help prevent widespread infection by P. salmonis, in addition to being able to be used as a booster after a primary vaccine to maintain high levels of circulating protective antibodies, greatly helping to reduce the economic losses caused by the pathogen.

Subcellular Location of Piscirickettsia salmonis Heat Shock Protein 60 (Hsp60) Chaperone by Using Immunogold Labeling and Proteomic Analysis.

Piscirickettsia salmonis is the causative bacterial agent of piscirickettsiosis, a systemic fish disease that significantly impacts the Chilean salmon industry. This bacterium possesses a type IV secretion system (T4SS), several proteins of the type III secretion system (T3SS), and a single heat shock protein 60 (Hsp60/GroEL). It has been suggested that due to its high antigenicity, the P. salmonis Hsp60 could be surface-exposed, translocated across the membrane, and (or) secreted into the extracellular matrix. This study tests the hypothesis that P. salmonis Hsp60 could be located on the bacterial surface. Immunogold electron microscopy and proteomic analyses suggested that although P. salmonis Hsp60 was predominantly associated with the bacterial cell cytoplasm, Hsp60-positive spots also exist on the bacterial cell envelope. IgY antibodies against P. salmonis Hsp60 protected SHK-1 cells against infection. Several bioinformatics approaches were used to assess Hsp60 translocation by the T4SS, T3SS, and T6SS, with negative results. These data support the hypothesis that small amounts of Hsp60 must reach the bacterial cell surface in a manner probably not mediated by currently characterized secretion systems, and that they remain biologically active during P. salmonis infection, possibly mediating adherence and (or) invasion.

Cytosolic phosphoenolpyruvate carboxykinase is expressed in α-cells from human and murine pancreas.

The pancreatic islets of Langerhans, mainly formed by glucagon-producing α-cells and insulin-producing ß-cells, are critical for glucose homeostasis. Insulin and glucagon oppositely modulate blood glucose levels in health, but a combined decline in insulin secretion together with increased glucagon secretion contribute to hyperglycemia in diabetes. Despite this bi-hormonal dysregulation, most studies have focused on insulin secretion and much less is known about glucagon secretion. Therefore, a deeper understanding of α-cell metabolism and glucagon secretion is of great interest. Here, we show that phosphoenolpyruvate carboxykinase (PCK1), an essential cataplerotic enzyme involved in metabolism and long considered to be absent from the pancreatic islet, is expressed in pancreatic α-cells of both murine and human. Furthermore, PCK1 transcription is induced by fasting and diabetes in rat pancreas, which indicates that the PCK1 activity is required for α-cell adaptation to different metabolic states. To our knowledge, this is the first evidence implicating PCK1 expression in α-cell metabolism.

Analysis of single nucleotide polymorphisms (SNPs) associated with antibiotic resistance genes in Chilean Piscirickettsia salmonis strains.

The aetiological agent of Piscirickettsiosis is Piscirickettsia salmonis, a Gram-negative intracellular pathogen, and high doses of antibiotics have regularly been employed to treat this infection. Seven florfenicol and/or oxytetracycline resistance genes (tet pump, tetE, Tclor/flor, Tbcr, TfloR, ompF and mdtN) were identified in strains by in silico genome analyses. Later, the number of single nucleotide polymorphisms (SNPs) and its relationship with the resistance to these antibiotics were identified and analysed, using the original LF-89 strain as reference. Trials to determine and compare the minimum inhibitory concentration (MIC) of oxytetracycline and florfenicol in each strain, as well as to quantify the gPCR transcripts levels in the selected genes, were performed. Therefore, variations in the resistance to both antibiotics were observed, where the strain with fewer SNPs showed the highest susceptibility. Consistently, the in silico 3D analyses of proteins encoded by the selected genes revealed structural changes, evident in the sequences with the highest number of SNPs. These results showed that the bacterial resistance to oxytetracycline was mainly linked to the presence of SNPs in relevant sites, antibiotic resistance genes and an OmpF porin, leading to important changes in the protein structure.

The Antidiabetic Agent Sodium Tungstate Induces Abnormal Glycogen Accumulation in Renal Proximal Tubules from Diabetic IRS2-Knockout Mice.

The kidney is an insulin-sensitive organ involved in glucose homeostasis. One major effect of insulin is to induce glycogen storage in the liver and muscle. However, no significant glycogen stores are detected in normal kidneys, but diabetic subjects present a characteristic renal histopathological feature resulting from extensive glycogen deposition mostly in nonproximal tubules. The mechanism of renal glycogen accumulation is yet poorly understood. Here, we studied in situ glycogen accumulation in the kidney from diabetic IRS2-knockout mice and the effect of the insulin-mimetic agent sodium tungstate (NaW). IRS2-knockout mice displayed hyperglycemia and hyperinsulinemia. NaW only normalized glycemia. There was no evident morphological difference between kidneys from untreated wild-type (WT), NaW-treated WT, and untreated IRS2-knockout mice. However, NaW-treated IRS2-knockout mice showed tubular alterations resembling clear cells in the cortex, but not in the outer medulla, that were correlated with glycogen accumulation. Immunohistochemical detection of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase, mostly expressed by renal proximal tubules, showed that altered tubules were of proximal origin. Our preliminary study suggests that IRS2 differentially regulates glycogen accumulation in renal tubules and that NaW treatment in the context of IRS2 ablation induces abnormal glycogen accumulation in cortical proximal tubules.

Comparative Genomic Analysis of Two Chilean Renibacterium salmoninarum Isolates and the Type Strain ATCC 33209T.

Renibacterium salmoninarum, a slow-growing facultative intracellular pathogen belonging to the high C + G content Actinobacteria phylum, is the causative agent of bacterial kidney disease, a progressive granulomatous infection affecting salmonids worldwide. This Gram-positive bacterium has existed in the Chilean salmonid industry for >30 years, but little or no information is available regarding the virulence mechanisms and genomic characteristics of Chilean isolates. In this study, the genomes of two Chilean isolates (H-2 and DJ2R) were sequenced, and a search was conducted for genes and proteins involved in virulence and pathogenicity, and we compare with the type strain ATCC 33209 T genome. The genome sizes of H-2 and DJ2R are 3,155,332 bp and 3,155,228 bp, respectively. They genomes presented six ribosomal RNA, 46 transcription RNA, and 25 noncodingRNA, and both had the same 56.27% G + C content described for the type strain ATCC 33209 T. A total of 3,522 and 3,527 coding sequences were found for H-2 and DJ2R, respectively. Meanwhile, the ATCC 33209 T type strain had 3,519 coding sequences. The in silico genome analysis revealed a genes related to tricarboxylic acid cycle, glycolysis, iron transport and others metabolic pathway. Also, the data indicated that R salmoninarum may have a variety of possible virulence-factor and antibiotic-resistance strategies. Interestingly, many of genes had high identities with Mycobacterium species, a known pathogenic Actinobacteria bacterium. In summary, this study provides the first insights into and initial steps towards understanding the molecular basis of antibiotic resistance, virulence mechanisms and host/environment adaptation in two Chilean R. salmoninarum isolates that contain proteins of which were similar to those of Mycobacterium. Furthermore, important information is presented that could facilitate the development of preventive and treatment measures against R. salmoninarum in Chile and worldwide.

In vitro genomic and proteomic evidence of a type IV pili-like structure in the fish pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is an intracellular γ-proteobacteria and the etiological agent of piscirickettsiosis, which causes massive economic losses in the Chilean salmon industry. The type IV pili (T4P) play an important role in adherence to host cell surfaces and bacterial pathogenicity. T4P contains a variable number of components, as predicted in P. salmonis genomes. However, no studies have determined if P. salmonis possesses T4P. The aims of this investigation were to identify T4P components in the P. salmonis type strain LF-89T, evaluate respective transcript expressions, and analyze the main putative T4P proteins using bioinformatics and proteomic approaches. Two main clusters of P. salmonis T4P genes were found. Expression of the pilA gene was upregulated at 4 h post-infection (hpi), while pilQ was upregulated 4 days post-infection. At 16 hpi, pilB and pilD were strongly upregulated. The PilA amino acid sequence analysis showed a conserved N-terminal domain and sequence motifs critical for T4P biosynthesis. MudPIT analysis revealed PilA in the P. salmonis LF-89T proteome, and TEM showed pili-like filamentous structures on the P. salmonis surface. These results strongly suggest the presence of a T4P-like structure in P. salmonis.

Core non-coding RNAs of Piscirickettsia salmonis.

Piscirickettsia salmonis, a fastidious Gram-negative intracellular facultative bacterium, is the causative agent o Piscirickettsiosis. P. salmonis has broad host range with a nearly worldwide distribution, causing significant mortality. The molecular regulatory mechanisms of P. salmonis pathogenesis are relatively unknown, mainly due to its difficult in vitro culture and genomic differences between genogroups. Bacterial non-coding RNAs (ncRNAs) are important post-transcriptional regulators of bacterial physiology and virulence that are predominantly transcribed from intergenic regions (trans-acting) or antisense strand of open reading frames (cis-acting). The repertoire of ncRNAs present in the genome of P. salmonis and its possible role in bacterial physiology and pathogenesis are unknown. Here, we predicted and analyzed the core ncRNAs of P. salmonis base on structure and correlate this prediction to RNA sequencing data. We identified a total of 69 ncRNA classes related to tRNAs, rRNA, thermoregulators, antitoxins, ribozymes, riboswitches, miRNAs and antisense-RNAs. Among these ncRNAs, 29 classes of ncRNAs are shared between all P. salmonis genomes, constituting the core ncRNAs of P. salmonis. The ncRNA core of P. salmonis could serve to develop diagnostic tools and explore the role of ncRNA in fish pathogenesis.

Comparative Pan-Genome Analysis of Piscirickettsia salmonis Reveals Genomic Divergences within Genogroups.

Piscirickettsia salmonis is the etiological agent of salmonid rickettsial septicemia, a disease that seriously affects the salmonid industry. Despite efforts to genomically characterize P. salmonis, functional information on the life cycle, pathogenesis mechanisms, diagnosis, treatment, and control of this fish pathogen remain lacking. To address this knowledge gap, the present study conducted an in silico pan-genome analysis of 19 P. salmonis strains from distinct geographic locations and genogroups. Results revealed an expected open pan-genome of 3,463 genes and a core-genome of 1,732 genes. Two marked genogroups were identified, as confirmed by phylogenetic and phylogenomic relationships to the LF-89 and EM-90 reference strains, as well as by assessments of genomic structures. Different structural configurations were found for the six identified copies of the ribosomal operon in the P. salmonis genome, indicating translocation throughout the genetic material. Chromosomal divergences in genomic localization and quantity of genetic cassettes were also found for the Dot/Icm type IVB secretion system. To determine divergences between core-genomes, additional pan-genome descriptions were compiled for the so-termed LF and EM genogroups. Open pan-genomes composed of 2,924 and 2,778 genes and core-genomes composed of 2,170 and 2,228 genes were respectively found for the LF and EM genogroups. The core-genomes were functionally annotated using the Gene Ontology, KEGG, and Virulence Factor databases, revealing the presence of several shared groups of genes related to basic function of intracellular survival and bacterial pathogenesis. Additionally, the specific pan-genomes for the LF and EM genogroups were defined, resulting in the identification of 148 and 273 exclusive proteins, respectively. Notably, specific virulence factors linked to adherence, colonization, invasion factors, and endotoxins were established. The obtained data suggest that these genes could be directly associated with inter-genogroup differences in pathogenesis and host-pathogen interactions, information that could be useful in designing novel strategies for diagnosing and controlling P. salmonis infection.

Nutritional Immunity Triggers the Modulation of Iron Metabolism Genes in the Sub-Antarctic Notothenioid Eleginops maclovinus in Response to Piscirickettsia salmonis.

Iron deprivation is a nutritional immunity mechanism through which fish can limit the amount of iron available to invading bacteria. The aim of this study was to evaluate the modulation of iron metabolism genes in the liver and brain of sub-Antarctic notothenioid Eleginops maclovinus challenged with Piscirickettsia salmonis. The specimens were inoculated with two P. salmonis strains: LF-89 (ATCC® VR-1361™) and Austral-005 (antibiotic resistant). Hepatic and brain samples were collected at intervals over a period of 35 days. Gene expression (by RT-qPCR) of proteins involved in iron storage, transport, and binding were statistically modulated in infected fish when compared with control counterparts. Specifically, the expression profiles of the transferrin and hemopexin genes in the liver, as well as the expression profiles of ferritin-M, ferritin-L, and transferrin in the brain, were similar for both experimental groups. Nevertheless, the remaining genes such as ferritin-H, ceruloplasmin, hepcidin, and haptoglobin presented tissue-specific expression profiles that varied in relation to the injected bacterial strain and sampling time-point. These results suggest that nutritional immunity could be an important immune defense mechanism for E. maclovinus against P. salmonis injection. This study provides relevant information for understanding iron metabolism of a sub-Antarctic notothenioid fish.

The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins.

Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.

In vitro expression of Sec-dependent pathway and type 4B secretion system in Piscirickettsia salmonis.

Piscirickettsia salmonis is an intracellular bacterium and the causative agent of Piscirickettsiosis, a disease responsible for considerable mortalities in the Chilean salmon farming industry. Currently, P. salmonis protein translocation across the membrane and the mechanisms by which virulence factors are delivered to host cells are poorly understood. However, it is known that Gram-negative bacteria possess several mechanisms that transport proteins to the periplasmic and extracellular compartments. The aim of this study was to evaluate the expressional changes of several genes in the P. salmonis Sec-dependent pathway and type 4B secretion system during in vitro infection. Genes homologous and the main proteins belonging to Sec-dependent pathway and Type 4 Dot/Icm secretion system were found in the genome and proteome of P. salmonis AUSTRAL-005 strain. Additionally, several genes of these protein transport mechanisms were overexpressed during in vitro P. salmonis infection in SHK-1 cell line. The obtained data indicate that the Sec-dependent pathway and Type 4B secretion system are biologically active during P. salmonis infection. These mechanisms could contribute to the recycling of proteins into the inner and outer bacterial membrane and in translocate virulence factors to infected cell, which would favor the structural integrity and virulence of this bacterium.

Membrane vesicles from Piscirickettsia salmonis induce protective immunity and reduce development of salmonid rickettsial septicemia in an adult zebrafish model.

Infections caused by the facultative intracellular bacterial pathogen Piscirickettsia salmonis remains an unsolved problem for the aquaculture as no efficient treatments have been developed. As a result, substantial amounts of antibiotic have been used to limit salmonid rickettsial septicemia (SRS) disease outbreaks. The antibiotic usage has not reduced the occurrence, but lead to an increase in resistant strains, underlining the need for new treatment strategies. P. salmonis produce membrane vesicles (MVs); small spherical structures know to contain a variety of bacterial components, including proteins, lipopolysaccharides (LPS), DNA and RNA. MVs mimics' in many aspects their mother cell, and has been reported as alternative vaccine candidates. Here, MVs from P. salmonis was isolated and evaluated as a vaccine candidate against SRS in an adult zebrafish infection model. When zebrafish was immunized with MVs they were protected from subsequent challenge with a lethal dose of P. salmonis. Histological analysis showed a reduced bacterial load upon challenge in the MV immunized group, and the mRNA expression levels of several immune related genes altered, including mpeg1.1, tnfα, il1b, il10 and il6. The MVs induced the secretion of IgM upon immunization, indicating an immunogenic effect of the vesicles. Taken together, the data demonstrate a vaccine potential of MVs against P. salmonis.

Molecular characterization of infectious pancreatic necrosis virus strains isolated from the three types of salmonids farmed in Chile.

BACKGROUND: The infectious pancreatic necrosis virus (IPNV) causes significant economic losses in Chilean salmon farming. For effective sanitary management, the IPNV strains present in Chile need to be fully studied, characterized, and constantly updated at the molecular level. METHODS: In this study, 36 Chilean IPNV isolates collected over 6 years (2006-2011) from Salmo salar, Oncorhynchus mykiss, and Oncorhynchus kisutch were genotypically characterized. Salmonid samples were obtained from freshwater, estuary, and seawater sources from central, southern, and the extreme-south of Chile (35° to 53°S). RESULTS: Sequence analysis of the VP2 gene classified 10 IPNV isolates as genogroup 1 and 26 as genogroup 5. Analyses indicated a preferential, but not obligate, relationship between genogroup 5 isolates and S. salar infection. Fifteen genogroup 5 and nine genogroup 1 isolates presented VP2 gene residues associated with high virulence (i.e. Thr, Ala, and Thr at positions 217, 221, and 247, respectively). Four genogroup 5 isolates presented an oddly long VP5 deduced amino acid sequence (29.6 kDa). Analysis of the VP2 amino acid motifs associated with clinical and subclinical infections identified the clinical fingerprint in only genogroup 5 isolates; in contrast, the genogroup 1 isolates presented sequences predominantly associated with the subclinical fingerprint. Predictive analysis of VP5 showed an absence of transmembrane domains and plasma membrane tropism signals. WebLogo analysis of the VP5 BH domains revealed high identities with the marine birnavirus Y-6 and Japanese IPNV strain E1-S. Sequence analysis for putative 25 kDa proteins, coded by the ORF between VP2 and VP4, exhibited three putative nuclear localization sequences and signals of mitochondrial tropism in two isolates. CONCLUSIONS: This study provides important advances in updating the characterizations of IPNV strains present in Chile. The results from this study will help in identifying epidemiological links and generating specific biotechnological tools for controlling IPNV outbreaks in Chilean salmon farming.

Consecutive emamectin benzoate and deltamethrin treatments affect the expressions and activities of detoxification enzymes in the rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) subjected to three consecutive, alternating treatments with emamectin benzoate (EMB) and deltamethrin (DM) during outbreaks of Caligus rogercresseyi in a farm located in southern Chile (Hornopiren, Chiloé), were studied to determine the effects of these treatments on the protein and enzymatic activity levels of cytochrome P450 1A (CYP1A), flavin-containing monooxygenase (FMO) and glutathione S-transferase (GST) in different tissues. Consecutive and alternating EMB/DM treatments resulted in a 10-fold increase and 3-fold decrease of CYP1A protein levels in the intestine and gills, respectively. Notably, CYP1A activity levels decreased in most of the analyzed tissues. FMO protein and activity levels markedly increased in the kidney and the intestine. GST was up-regulated in all tissues, either as protein or enzyme activity. When comparing consecutive EMB/DM treatments against previous studies of EMB treatment alone, CYP1A activity levels were similarly diminished, except in muscle. Likewise, FMO activity levels were increased in most of the analyzed tissues, particularly in the muscle, kidney, and intestine. The increases observed for GST were essentially unchanged between consecutive EMB/DM and EMB only treatments. These results indicate that consecutive EMB/DM treatments in rainbow trout induce the expression and activity of FMO and GST enzymes and decrease CYP1A activity. These altered activities of detoxification enzymes could generate imbalances in metabolic processes, synthesis, degradation of hormones and complications associated with drug interactions. It is especially important when analyzing possible effects of consecutive antiparasitic treatments on withholding periods and salmon farming yields.