Prevalence,geographic distribution and phenotypic differences of Piscirickettsia salmonis EM‐90‐like isolates

Early reports accounted for two main genotypes of Piscirickettsia salmonis, a fish pathogen and causative agent of piscirickettsiosis, placing the single isolate EM‐90 apart from the prototypic LF‐89 and related isolates. In this study, we provide evidence that, contrary to what has been supposed, the EM‐90‐like isolates are highly prevalent and disseminated across Chilean marine farms. Molecular analysis of 507 P. salmonis field isolates derived from main rearing areas, diverse hosts and collected over 6 years, revealed that nearly 50% of the entire collection were indeed typed as EM‐90‐like. Interestingly, these isolates showed a marked host preference, being recovered exclusively from Atlantic salmon (Salmo salar) samples. Although both strains produce undistinguishable pathological outcomes, differences regarding growth kinetics and susceptibility to the antibiotics and bactericidal action of serum could be identified. In sum, our results allow to conclude that the EM‐90‐like isolates represent an epidemiologically relevant group in the current situation of piscirickettsiosis. Based on the consistency between genotype and phenotype exhibited by this strain, we point out the need for genotypic studies that may be as important for the Chilean salmon industry as the continuous surveillance of antimicrobial susceptibility patterns.     

Comparative evaluation of experimental challenge by intraperitoneal injection and cohabitation of Atlantic salmon (Salmo salar L) after vaccination against Piscirickettsia salmonis (EM90‐like)

The Chilean aquaculture has been challenged for years by piscirickettsiosis. A common prophylactic measurement to try to reduce the impact from this disease is vaccination, but the development of vaccines that induce satisfactory protection of the fish in the field has so far not been successful. Experimental challenge models are used to test vaccine efficacy. The aim of this study was to evaluate the performance of experimental vaccines after challenge by the two most widely used challenge routes, intraperitoneal injection and cohabitation. A total of 1,120 Atlantic salmon were vaccinated with non‐commercial experimental vaccines with increasing amounts of an inactivated Piscirickettsia salmonis EM90‐like isolate. Differences in mortality, macroscopic and microscopic pathological changes, bacterial load and immune gene expression were compared after challenge by different routes. The results revealed a similar progression of the diseases after challenge by both routes and no gross differences reflecting the efficacy of the vaccines could be identified. The analysis of the immune genes suggests a possible suppression of the cellular immunity by CD8 T cell and with this stimulation of bacterial survival and replication. Comparative studies of experimental challenge models are valuable with regard to identifying the best model to mimic real‐life conditions and vaccines’ performance.     

The mutagenesis of a type IV secretion system locus of Piscirickettsia salmonis leads to the attenuation of the pathogen in Atlantic salmon,Salmo salar

Piscirickettsiosis is a threatening infectious disease for the salmon industry, due to it being responsible for significant economic losses. The control of outbreaks also poses considerable environmental challenges. Despite Piscirickettsia salmonis having been discovered as the aetiological agent of the disease more than 25 years ago, its pathogenicity remains poorly understood. Among virulence factors identified so far, type four secretion systems (T4SS) seem to play a key role during the infection caused by the bacterium. We report here the genetic manipulation of P. salmonis by means of the transference of plasmid DNA in mating assays. An insertion cassette was engineered for targeting the icmB gene, which encodes a putative T4SS‐ATPase and is carried by one of the chromosomal T4SS clusters found within the genome of P. salmonis PM15972A1, a virulent representative of the EM‐90‐like strain. The molecular characterization of the resulting mutant strain demonstrated that the insertion interrupted the target gene. Further in vitro testing of the icmB mutant showed a dramatic drop in infectivity as tested in CHSE‐214 cells, which is in agreement with its attenuated behaviour observed in vivo. Altogether, our results demonstrate that, similar to other facultative intracellular pathogens, P. salmonis’ virulence relies on an intact T4SS.     

Identification of genomic islands in Chilean Piscirickettsia salmonis strains and analysis of gene expression involved in virulence

Piscirickettsia salmonis, an agent of Piscirickettsiosis, is the cause of major losses in the Chilean salmon industry. We identified, characterized and bioinformatically analysed genomic islands in field strains of P. Salmonis, using the bioinformatic software PIPS, that uses the characteristics of the islands of pathogenicity to identify them. We analysed nine partially sequenced genomes in different new field strains, and compared them with the LF‐89 (Type strain) genome, selecting a genomic island present in all of them. We then evaluated the relative expression of three genes present in that island. From the obtained results, we conclude that the expression of the tcf gene is directly proportional to the cytopathogenicity in vitro of the bacteria; the product of the dnsa gene could contribute to its pathogenicity, but would be potentiated by one or more factors. The product of the gene liso is necessary for the virulence process and could have functions in early stages of infection. Regarding the strains, the IBM‐040 strain showed a significant increase in the expression of all the genes in the study. Contrarily, LF‐89 only presented a significant increase in expression of the gene liso, which correlates with the cytopathogenicity in vitro observed in the SHK‐1 cells.     

Development of piscirickettsiosis in Atlantic salmon (Salmo salar L.) smolts after intraperitoneal and cohabitant challenge using an EM90‐like isolate: A comparative study

Piscirickettsiosis, caused by the intracellular Gram‐negative bacteria Piscirickettsia salmonis, is at present the most devastating disease in the Chilean salmon industry. The aim of this study was to analyse disease development after challenge with a P. salmonis strain (EM90‐like) under a controlled environment by comparing intraperitoneal challenge with cohabitation challenge. The P. salmonis EM90‐like isolate was cultured in a liquid medium for the challenge of 400 Atlantic salmon (Salmo salar) smolts. Cumulative mortality was registered, necropsy was performed, and bacterial distribution in the tissues and histopathological changes were analysed. The results revealed a similar progression of the disease for the two different challenge models. Pathological and histopathological changes became more visible during the development of the clinical phase of the disease. Bacterial DNA was identified in all the analysed tissues indicating a systemic infection. Bacterial tropism to visceral organs was demonstrated by real‐time quantitative PCR and immunohistochemistry. Better knowledge of disease development during P. salmonis infection may contribute to further development of challenge models that mimic the field situation during piscirickettsiosis outbreaks. The models can be used to develop and test future preventive measures against the disease.     

In vivo growth and genomic characterization of rickettsia‐like organisms isolated from farmed Chinook salmon (Oncorhynchus tshawytscha) in New Zealand

A rickettsia‐like organism, designated NZ‐RLO2, was isolated from Chinook salmon (Oncorhynchus tshawytscha) farmed in the South Island, New Zealand. In vivo growth showed NZ‐RLO2 was able to grow in CHSE‐214, EPC, BHK‐21, C6/36 and Sf21 cell lines, while Piscirickettsia salmonis LF‐89^T grew in all but BHK‐21 and Sf21. NZ‐RLO2 grew optimally in EPC at 15°C, CHSE‐214 and EPC at 18°C. The growth of LF‐89 ^T was optimal at 15°C, 18°C and 22°C in CHSE‐24, but appeared less efficient in EPC cells at all temperatures. Pan‐genome comparison of predicted proteomes shows that available Chilean strains of P. salmonis grouped into two clusters (p‐value = 94%). NZ‐RLO2 was genetically different from previously described NZ‐RLO1, and both strains grouped separately from the Chilean strains in one of the two clusters (p‐value = 88%), but were closely related to each other. TaqMan and Sybr Green real‐time PCR targeting RNA polymerase (rpoB) and DNA primase (dnaG), respectively, were developed to detect NZ‐RLO2. This study indicates that the New Zealand strains showed a closer genetic relationship to one of the Chilean P. salmonis clusters; however, more Piscirickettsia genomes from wider geographical regions and diverse hosts are needed to better understand the classification within this genus.     

Disruption of host‐seeking behaviour by the salmon louse,Lepeophtheirus salmonis,using botanically derived repellents

The potential for developing botanically derived natural products as novel feed‐through repellents for disrupting settlement of the salmon louse, Lepeophtheirus salmonis (Caligidae) upon farmed Atlantic salmon, Salmo salar, was investigated using an established laboratory vertical Y‐tube behavioural bioassay for assessing copepodid behaviour. Responses to artificial sea water conditioned with the odour of salmon, or to the known salmon‐derived kairomone component, α‐isophorone, in admixture with selected botanical materials previously known to interfere with invertebrate arthropod host location were recorded. Materials included oils extracted from garlic, Allium sativum (Amaryllidaceae), rosemary, Rosmarinus officinalis (Lamiaceae), lavender, Lavandula angustifolia (Lamiaceae), and bog myrtle, Myrica gale (Myricaceae), and individual components (diallyl sulphide and diallyl disulphide from garlic; allyl, propyl, butyl, 4‐pentenyl and 2‐phenylethyl isothiocyanate from plants in the Brassica genus). Removal of attraction to salmon‐conditioned water (SCW) or α‐isophorone was observed when listed materials were presented at extremely low parts per trillion (ppt), that is picograms per litre or 10^?12 level. Significant masking of attraction to SCW was observed at a level of 10 ppt for diallyl disulphide and diallyl sulphide, and allyl isothiocyanate and butyl isothiocyanate. The potential of very low concentrations of masking compounds to disrupt Le. salmonis copepodid settlement on a host fish has been demonstrated in vitro.     

Cloning,characterization, expression analysis and RNAi of Retinoblastoma‐like gene from black tiger shrimp (Penaeus monodon)

Retinoblastoma (Rb) is a multifunctional regulator involved in several key cellular processes, such as cell cycle control, cell differentiation, tumorigenesis and senescence. In this study, an Rb‐like gene, PmRBL, was cloned from black tiger shrimp (Penaeus monodon). The full‐length cDNA sequence of PmRBL is 4,069 bp with an open reading frame of 3,243 bp, which encodes 1,080 amino acids. Quantitative real‐time PCR (qRT‐PCR) analysis indicated that PmRBL was highly expressed in the gills, hepatopancreas and ovaries of P. monodon. The highest PmRBL expression levels were observed in stage III of the ovarian development in P. monodon. RNA interference experiments were conducted to examine the expression profiles of PmRBL, PmCDK2 and PmCyclin E. The knockdown of PmRBL in the ovary and hepatopancreas by dsRNA‐RBL was successful. After dsRNA‐RBL was injected into the shrimp, the relative expression levels of PmCDK2 and PmCyclin E were upregulated at 12–72 hr in the ovaries and hepatopancreas. The localization and level of PmRBL expression in the ovary and hepatopancreas were investigated through in situ hybridization, which revealed consistent results with those of qRT‐PCR. Therefore, PmRBL, PmCDK2 and PmCyclin E may be involved in vitellogenin synthesis and ovarian maturation in P. monodon.     

Effects of dietary astaxanthin on growth,blood biochemistry,antioxidant, immune and inflammatory response in lipopolysaccharide‐challenged Channa argus

The present study investigated the effects of dietary astaxanthin on the growth, blood biochemical, antioxidant, immune and inflammatory response in lipopolysaccharide‐challenged Channa argus. A total of 0, 50, 100 and 200 mg/kg of astaxanthin were added to the basal die for 56 days. After the feeding experiment, each group was subjected to a lipopolysaccharide challenge (except for the Control group). The results showed that adding astaxanthin to the diet can significantly increase the weight gain and specific growth rate and decrease the feed conversion ratio of C. argus; the highest weight gain, specific growth rate and minimum feed conversion ratio occurred in the 100 mg/kg group. Furthermore, dietary astaxanthin supplementation can alleviate the negative effects of lipopolysaccharides by increasing the levels of alkaline phosphatase, lysozyme, complement 3, complement 4, total serum protein, albumin, globulin, superoxide dismutase, catalase and glutathione peroxidase and decrease the serum cortisol, aspartate aminotransferase, alanine aminotransferase, glutathione peroxidase, and malondialdehyde. Dietary astaxanthin supplementation also can decrease the relative expression of inflammatory genes (nuclear factor κB, interleukin‐1, interleukin‐8 and tumour necrosis factor‐α) in the liver, spleen, kidney and intestine. To summarise, dietary astaxanthin addition can improve the growth performance and attenuate the negative effects of lipopolysaccharide challenge in C. argus. The optimal amount of astaxanthin is 100 mg/kg.     

The immune response in rohu,Labeo rohita (Actinopterygii: Cyprinidae) to Argulus siamensis (Branchiura: Argulidae) infection: kinetics of immune gene expression and innate immune response

Argulus siamensis is a major pathogen in freshwater aquaculture. The immune responses of Indian major carp, Labeo rohita to experimental infection of A. siamensis was evaluated by quantitation of immune‐relevant gene expression in head kidney and skin, and serum innate immune parameters through the course of infection. In skin of infected fish, antioxidant genes like natural killer cell enhancing factor (NKEF‐B) and superoxide dismutase (MnSOD) were significantly up‐regulated in addition to lysozyme G and β₂ microglobulin (β₂M). Both tumour necrosis factor α (TNFα) and toll‐like receptor 22 (TLR22) genes were significantly down‐regulated in skin during early phases of the infection. Most of the genes exhibited significant down‐regulation in head kidney; immunoglobulin (IgM) and β₂M genes being the exceptions which were significantly up‐regulated at 12 h and 3 days post infection. Most of the innate immune parameters like serum complement activity and ceruloplasmin levels showed significant reduction in infected fish. The observed results are indicative of A. siamensis modulating the immune response of rohu by down‐regulation of many immune factors which may explain the susceptibility of rohu to A. siamensis infection. The interaction of this parasite with the host need to be further explored to understand its pathogenesis.     

Pro‐inflammatory caspase‐1 activation during the immune response in cells from rainbow trout Oncorhynchus mykiss (Walbaum 1792) challenged with pathogen‐associated molecular patterns

In response to pathogens, the higher vertebrate innate immune system activates pro‐inflammatory caspase‐1 which is responsible for the processing and secretion of several important cytokines involved in the host's defence against infection. To date, caspase‐1 has been described in few teleost fish, and its activity has been demonstrated through substrate cleavage and inhibition by pharmacological agents. In this study, the detection of the active form of caspase‐1 during the immune response in salmonid fish is described, where two antibodies were produced. These antibodies differentially recognize the structural epitopes of the inactive pro‐caspase‐1 and the processed active form of the caspase. Firstly, caspase‐1 activation was demonstrated in vitro by ELISA, Western blotting and immunocytochemistry in rainbow trout macrophages exposed to different pathogen‐associated molecular patterns plus the pathogen Aeromonas hydrophila. This activity was clearly abrogated by a caspase inhibitor and seems to be unrelated to IL‐1β secretion. Caspase‐1 activation was then validated in vivo in gill cells from fish challenged with Aeromonas salmonicida. These results represent the first demonstration of caspase‐1 activation in salmonids, and the first evidence of the putative regulatory role which this protease plays in inflammatory response in this fish group, as described for some other teleosts and mammals.     

Effects of dietary live or heat‐inactivated autochthonous Bacillus pumilus SE5 on growth performance,immune responses and immune gene expression in grouper Epinephelus coioides

To evaluate the possible dietary application of live and heat‐inactivated probiotic Bacillus pumilus SE5 in grouper Epinephelus coioides, juveniles (14.6 ± 0.2 g) were fed either a basal control diet (without probiotic) or the basal diet supplemented with 1.0 × 10⁸ CFU g^?1 live (T1) and heat‐inactivated B. pumilus SE5 (T2). The heat‐inactivated probiotic significantly improved the final weight, weight gain (WG) and specific growth rate (SGR) at day 60 and significantly decreased the feed conversion ratio (FCR) at day 30 and 60, while the viable probiotic significantly decreased the FCR at day 60 (P < 0.05). Phagocytic activity, serum complement C3 and IgM levels as well as SOD activity elevated significantly in fish fed the heat‐inactivated probiotic for 60 days (P < 0.05). Furthermore, the heat‐inactivated probiotic remarkably up‐regulated expression of TLR2 and pro‐inflammatory cytokines (IL‐8 and IL‐1β) in head kidney (P < 0.05), but the viable probiotic failed to do so. These results indicated that heat‐inactivated B. pumilus SE5 can effectively improve the growth performance and immune responses of E. coioides.     

How are the salmon lice (Lepeophtheirus salmonis Krøyer, 1837) in Atlantic salmon farming affected by different control efforts: A case study of an intensive production area with coordinated production cycles and changing delousing practices in 2013–2018

The aims of the present study were to describe the salmon lice (Lepeophtheirus salmonis Krøyer, 1837) situation in an intensive salmon production area in mid‐Norway and to consider implications of changing practices of how salmon lice infestation can be controlled. The results in this study suggest that there are steps that can be carried out to keep salmon lice under control even during years when the temperature facilitates a quick salmon lice development. The present work indicates that the use of cleaner fish can delay the time it takes adult female lice to reach 0.1 per salmon in the beginning of a production cycle. It suggests that the timing of cleaner fish deployment into salmon cages can influence its effectiveness in controlling salmon lice. It also gives caution to letting salmon lice develop unchecked, even at levels far below the current lice limit, because of the difficulties to control salmon lice when the external infection pressure is too high. This study took place during a rapid change in delousing methods, in an area with coordinated salmon production. Despite its exploratory nature, this study offers insights into the salmon lice fluctuations in relation to efforts aimed at controlling it.     

The Three‐spined Stickleback,Gasterosteus aculeatus Linnaeus 1758, plays a minor role as a host of Lepeophtheirus salmonis (Krøyer 1837) in the Gulf of Maine

The sea louse, Lepeophtheirus salmonis (Krøyer 1837), is a significant parasite of farmed salmon throughout the Northern Hemisphere. Management of on‐farm louse populations can be improved by understanding the role that wild fish play in sustaining and providing refuge for the local population of sea lice. In this study, 1,064 sticklebacks were captured. Of these animals, 176 individuals were carrying a total of 238 sea lice, yielding a prevalence and intensity of 16.5% and 1.4 lice per fish, respectively. Detailed examination of the sea lice on the three‐spined sticklebacks captured in Cobscook Bay found two L. salmonis individuals using three‐spined sticklebacks as hosts. A 2012 survey of wild fish in Cobscook Bay, Maine, found multiple wild hosts for Caligus elongatus (von Nordmann 1832), including three‐spined sticklebacks (Gasterosteus aculeatus L.), but no L. salmonis were found in this earlier study.     

Label‐free quantification proteomics reveals the effects of dietary fish oil and soybean oil on the immune response of Chinese mitten crab,Eriocheir sinensis

The replacement of fish oil (FO) in Eriocheir sinensis can significantly reduce the cost of E. sinensis cultivation, while several studies have indicated that replacing FO with soybean oil (SO) could significantly reduce the resistance of E. sinensis to disease. However, the molecular mechanisms underlying these effects remain poorly understood. In this study, crabs were fed two diets containing FO or SO, following which a label‐free quantification proteomic analysis was employed. And the activity of enzymes involved in the nonspecific immune response was also measured. Growth performance was undifferentiated between the crabs fed with FO and SO. A total of 519 proteins were identified, and 70 proteins were significantly altered between the crabs fed the two different diets. Five proteins related to the immune response were identified to be differently expressed. C‐type lectin, haemocyanin subunit 6 and cryptocyanin were significantly downregulated, while fatty acid‐binding protein and catalase were highly expressed in the crabs fed SO. The activities of acid phosphatase, alkaline phosphatase, superoxide dismutase and phenoloxidase were all significantly changed in crab fed with different diets. These findings will provide novel insight into the molecular mechanism regarding the replacement of FO on the immune response of E. sinensis and provide evidences for the relationship between nutrition and immunity in E. sinensis.