Evidence of exotoxin secretion of Piscirickettsia salmonis,the causative agent of piscirickettsiosis

Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a disease which affects a variety of teleost species and that is particularly severe in salmonid fish. Bacterial‐free supernatants, obtained from cultures of three isolates of Piscirickettsia salmonis, were inoculated in Atlantic salmon, Salmo salar L., and in three continuous cell lines in an effort to determine the presence of secretion of extracellular products (ECPs) by this microorganism. Although steatosis was found in some liver samples, no mortalities or clinical signs occurred in the inoculated fish. Clear cytotoxicity was observed after inoculation in the cell lines CHSE‐214 and ASK, derived from salmonid tissues, but not in MDBK, which is of mammalian origin. The degree of cytotoxicity of the ECPs was different among the P. salmonis isolates tested. The isolate that evidenced the highest cytotoxicity in its ECPs exhibited only an intermediate virulence level after challenging fish with bacterial suspensions of the three P. salmonis isolates. Almost complete inhibition of the cytotoxic activity of ECPs was seen after proteinase K treatment, indicating their peptidic nature, and a total preclusion of the cytotoxicity was shown after their incubation at 50 °C for 30 min. Results show that P. salmonis can produce ECPs and at least some of them are thermolabile exotoxins that probably play a role in the pathogenesis of piscirickettsiosis.     

Piscirickettsiosis and Piscirickettsia salmonis in fish: a review

The bacterium Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis a severe disease that has caused major economic losses in the aquaculture industry since its appearance in 1989. Recent reports of P. salmonis or P. salmonis‐like organisms in new fish hosts and geographical regions have increased interest in the bacterium. Because this gram‐negative bacterium is still poorly understood, many relevant aspects of its life cycle, virulence and pathogenesis must be investigated before prophylactic procedures can be properly designed. The development of effective control strategies for the disease has been limited due to a lack of knowledge about the biology, intracellular growth, transmission and virulence of the organism. Piscirickettsiosis has been difficult to control; the failure of antibiotic treatment is common, and currently used vaccines show variable long‐term efficacy. This review summarizes the biology and characteristics of the bacterium, including its virulence; the infective strategy of P. salmonis for survival and evasion of the host immune response; the host immune response to invasion by this pathogen; and newly described features of the pathology, pathogenesis, epidemiology and transmission. Current approaches to the prevention of and treatment for piscirickettsiosis are discussed.     

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.     

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.     

Multilocus sequence typing detects new Piscirickettsia salmonis hybrid genogroup in Chilean fish farms: Evidence for genetic diversity and population structure

Piscirickettsia salmonisis the causative bacterial pathogen of piscirickettsiosis, a salmonid disease that causes notable mortalities in the worldwide aquaculture industry. Published research describes the phenotypic traits, virulence factors, pathogenicity and antibiotic‐resistance potential for various P. salmonisstrains. However, evolutionary and genetic information is scarce for P. salmonis. The present study used multilocus sequence typing (MLST) to gain insight into the population structure and evolution of P. salmonis. Forty‐two Chilean P. salmonisisolates, as well as the type strain LF‐89^T, were recovered from diseased Salmo salar, Oncorhynchus kisutchand Oncorhynchus mykissfrom two Chilean Regions. MLST assessed the loci sequences of dnaK, efp, fumC, glyA, murG, rpoD and trpB. Bioinformatics analyses established the genetic diversity among P. salmonis isolates (H = 0.5810). A total of 23 sequence types (ST) were identified, 53.48% of which were represented by ST1, ST5 and ST2. Population structure analysis through polymorphism patterns showed few polymorphic sites (218 nucleotides from 4,010 bp), while dN/dS ratio analysis indicated purifying selection for dnaK, epf, fumC, murG, and rpoD but neutral selection for the trpB loci. The standardized index of association indicated strong linkage disequilibrium, suggesting clonal population structure. However, recombination events were detected in a group of seven isolates. Findings included genogroups homologous to the LF‐89^T and EM‐90 strains, as well as a seven‐isolate hybrid genogroup recovered from both assessed regions (three O. mykiss and four S. salar isolates). The presented MLST scheme has comparative potential, with promising applications in studying distinct P. salmonis isolates (e.g., from different hosts, farms, geographical areas) and in understanding the epidemiology of this pathogen.     

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.     

Piscirickettsia salmonis infection in cultured lumpfish (Cyclopterus lumpus L.)

A Piscirickettsia salmonis infection was diagnosed in lumpfish (Cyclopterus lumpus L.) juveniles held in a marine research facility on the west coast of Ireland. The main clinical signs and pathology included marked ascites, severe multifocal liver necrosis and severe diffuse inflammation and necrosis of the exocrine pancreas and peri‐pancreatic adipose tissue. Numerous Piscirickettsia‐like organisms were observed by histopathology in the affected organs, and the bacterial species was characterized by molecular analysis. Sequencing of the partial 16S rDNA gene and internal transcribed spacer region showed the lumpfish sequences to be closely related to previously identified Atlantic salmon (Salmo salar L.) sequences from Ireland. To the authors’ knowledge, this is the first detection of P. salmonis in lumpfish worldwide. The infection is considered potentially significant in terms of lumpfish health and biosecurity.     

Comprehensive antibiotic susceptibility profiling of Chilean Piscirickettsia salmonis field isolates

Antibiotics have been extensively used against infections produced by Piscirickettsia salmonis, a fish pathogen and causative agent of piscirickettsiosis and one of the major concerns for the Chilean salmon industry. Therefore, the emergence of resistant phenotypes is to be expected. With the aim of obtaining a landscape of the antimicrobial resistance of P. salmonis in Chile, the susceptibility profiles for quinolones, florfenicol and oxytetracycline (OTC) of 292 field isolates derived from main rearing areas, different hosts and collected over 5 years were assessed. The results allowed for the determination of epidemiological cut‐off values that were used to characterize the pathogen population. This work represents the first large‐scale field study addressing the antimicrobial susceptibility of P. salmonis, providing evidence of the existence of resistant types with a high incidence of resistance to quinolones. Remarkably, despite the amounts and frequency of therapies, our results disclosed that the issue of resistance to florfenicol and OTC is still in the onset.     

Development of a Piscirickettsia salmonis immersion challenge model to investigate the comparative susceptibility of three salmon species

Piscirickettsia salmonis, the aetiological agent of salmonid rickettsial septicaemia (SRS), is a global pathogen of wild and cultured marine salmonids. Here, we describe the development and application of a reproducible, standardized immersion challenge model to induce clinical SRS in juvenile pink (Oncorhynchus gorbuscha), Atlantic (Salmo salar) and sockeye salmon (O. nerka). Following a 1‐hr immersion in 10⁵ colony‐forming units/ml, cumulative mortality in Atlantic salmon was 63.2% while mortality in sockeye salmon was 10%. Prevalence and levels of the bacterium in kidney prior to onset of mortality were lower in sockeye compared with Atlantic or pink salmon. The timing and magnitude of bacterial shedding were estimated from water samples collected during the exposure trials. Shedding was estimated to be 82‐fold higher in Atlantic salmon as compared to sockeye salmon and peaked in the Atlantic salmon trial at 36 d post‐immersion. These data suggest sockeye salmon are less susceptible to P. salmonis than Atlantic or pink salmon. Finally, skin lesions were observed on infected fish during all trials, often in the absence of detectable infection in kidney. As a result, we hypothesize that skin is the primary point of entry for P. salmonis during the immersion challenge.     

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.     

Growth characteristics of the intracellular pathogen,Piscirickettsia salmonis,in tissue culture and cell‐free media

Piscirickettsia salmonis is an intracellular bacterium that was first isolated and identified in fish cells. Several types of cell lines have been explored for their ability to provide the bacterium with a host cell to replicate in. Tissue culture has been used for growth and cultivation for nearly two decades, until the facultative nature of P. salmonis was confirmed upon the development of blood‐ and cysteine‐based agar. Since then, research has continued to drive the creation of novel agar and broth formulations in order to improve the efficacy of cultivation of P. salmonis. Until now, the techniques and components used for growth have not been thoroughly discussed. In this review, the methods and formulations for growth of P. salmonis in tissue culture and cell‐free media will be examined.     

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.