Salmonid Rickettsial Septicemia Research Articles

Successive Oral Immunizations Against Piscirickettsia Salmonis and Infectious Salmon Anemia Virus are Required to Maintain a Long-Term Protection in Farmed Salmonids.

Currently, there is a growing demand to determine the protective status of vaccinated fish in order to prevent diseases outbreaks. A set of different parameters that include the infectious and immunological status of vaccinated salmonids from 622 Chilean farms were analyzed during 2011–2014. The aim of this study was to optimize the vaccination program of these centers through the determination of the protective state of vaccinated fish using oral immunizations. This state was determined from the association of the concentration of the immunoglobulin M (IgM) in the serum and the mortality rate of vaccinated fish. Salmonids were vaccinated with different commercial mono- or polyvalent vaccines against salmonid rickettsial septicemia (SRS) and infectious salmon anemia (ISA), first by the intraperitoneal injection of oil-adjuvanted antigens and then by the stimulation of mucosal immunity using oral vaccines as a booster vaccination. The results showed that high levels of specific IgM antibodies were observed after injectable vaccination, reaching a maximum concentration at 600–800 degree-days. Similar levels of antibodies were observed when oral immunizations were administrated. The high concentration of antibodies [above 2750 ng/mL for ISA virus (ISAv) and 3500 ng/mL for SRS] was maintained for a period of 800 degree-days after each vaccination procedure. In this regard, oral immunizations maintained a long-term high concentration of anti-SRS and anti-ISAv specific IgM antibodies. When the concentration of antibodies decreased below 2000 pg/mL, a window of susceptibility to SRS infection was observed in the farm, suggesting a close association between antibody levels and fish protective status. These results demonstrated that, in the field, several oral immunizations are essential to uphold a high level of specific anti-pathogens antibodies and, therefore, the protective status during the whole productive cycle.

Identification of the clpB and bipA genes and an evaluation of their expression as related to intracellular survival for the bacterial pathogen Piscirickettsia salmonis

Piscirickettsia salmonis is the pathogen responsible for salmonid rickettsial septicemia (SRS), a disease that affects a wide variety of marine cultivated fish species and causes economic losses for the aquaculture industry worldwide. Many in vitro studies have reported on the capacity of this microorganism to replicate in the interior of cytoplasmic vesicles from varied fish cell lines. However, the mechanisms used by this bacteria to survive, replicate, and propagate in cell lines, especially in macrophages and monocytes, are unknown. A number of studies have described the diverse proteins in pathogens such as Legionella pneumophila, Coxiella burnetii, and Francisella tularensis which allow these to evade the cellular immune response and replicate in the interior of macrophages in different hosts. Some of these proteins are the virulence factor BipA/TypA and the heat shock protein ClpB, both of which have been widely characterized. The results of the current study present the complete coding sequence of the genes clpB and bipA from the P. salmonis genome. Moreover, the experimental results suggest that during the infectious process of the SHK-1 cellular line in P. salmonis, the pathogen significantly increases the expression of proteins ClpB and BipA. This would permit the pathogen to adapt to the hostile conditions produced by the macrophage and thus evade mechanisms of cellular degradation while facilitating replication in the interior of this salmon cell line.

Polymorphism in gyrA is associated to quinolones resistance in Chilean Piscirickettsia salmonis field isolates.

Keywords: antibiotic resistance, gyrA, Piscirickettsiasalmonis, quinolones.Piscirickettsia salmonis is a gram-negative, pleomor-phic coccoid-shaped and facultative intracellularfish pathogenic bacterium, that causes piscirickettsi-osis, a disease also known as salmonid rickettsialsepticaemia (SRS) (Fryer et al. 1990). In Chile,P. salmonis was isolated for the first time in 1989,but probably has been present since 1983 (Bravo C Rozas &Enriquez 2013). SRS has also been reported inimportant salmon producing countries such asCanada, Ireland, Scotland and Norway, but with alower economic impact (Rozas & Enriquez 2013).P. salmonis cells are slow growing and hard to culti-vate in free-cell media, which has hindered its isola-tion and further characterization. However, recentlydescribed supplemented media (Mikalsen et al.2008) facilitate their growth and the antibiotictesting assays to evaluate effective antimicrobial ther-apy. In this regard, quinolones were the former mostwidely used antimicrobial compounds in Chileanaquaculture, especially as a first line drug againstSRS.In Chile, a large proportion of antibiotics is usedfor prophylaxis rather than for chemotherapy. Forinstance, in comparison with Norway and Canada,1500 and eight times more antimicrobials wereneeded to obtain similar amounts of salmon inChile in 2007 (Cabello et al. 2013). The same year,930 metric tons of antibiotics were mostly used insalmon production. Of those antibiotics, 24% werequinolones (Cabello et al. 2013). This practice hasalready altered the microbiota of sediments in closeproximity to aquaculture sites, where a significantfraction of isolated bacteria is antibiotic resistant(Buschmann et al. 2012). Therefore, the emer-gence and potential spread of antibiotic-resistantP. salmonis isolates cannot be ruled out.Twenty P. salmonis isolates cultured from kid-ney samples of diseased fish collected from diversegeographic origin (Fig. 1) between 2010 and2012 were phenotypically characterized for quinol-ones susceptibility. The isolates were grown onagar (Mikalsen et al. 2008), at 18 °C for 10 daysand kept frozen in liquid nitrogen. For antibioticsensitivity testing, the minimal inhibitory concen-tration (MIC) for enrofloxacin, flumequine andoxolinic acid were assessed according to theinstructions given by the Clinical and LaboratoryStandards Institute (CLSI), guide M49-A. 96-wellmicroplates containing twofold serial dilutions(0.03–64 lgmL

Broth microdilution protocol for minimum inhibitory concentration (MIC) determinations of the intracellular salmonid pathogen Piscirickettsia salmonis to florfenicol and oxytetracycline

Journal of Fish DiseasesVolume 37, Issue 5 p. 505-509 Short Communication Broth microdilution protocol for minimum inhibitory concentration (MIC) determinations of the intracellular salmonid pathogen Piscirickettsia salmonis to florfenicol and oxytetracycline A J Yáñez, Corresponding Author A J Yáñez Facultad de Ciencias, Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, Chile Interdisciplinary Center for Aquaculture Research (INCAR), Concepción, ChileCorrespondence A J Yáñez, R Avendaño-Herrera and J G Carcamo, Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción, Chile (e-mails: ayanez@uach.cl, gcarcamo@uach.cl, ravendano@unab.cl) Search for more papers by this authorK Valenzuela, K Valenzuela Facultad de Ciencias, Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, ChileSearch for more papers by this authorC Matzner, C Matzner Facultad de Ciencias, Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, ChileSearch for more papers by this authorV Olavarría, V Olavarría Facultad de Ciencias, Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, ChileSearch for more papers by this authorJ Figueroa, J Figueroa Facultad de Ciencias, Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, Chile Interdisciplinary Center for Aquaculture Research (INCAR), Concepción, ChileSearch for more papers by this authorR Avendaño-Herrera, Corresponding Author R Avendaño-Herrera Interdisciplinary Center for Aquaculture Research (INCAR), Concepción, Chile Laboratorio de Patología de Organismos Acuáticos y Biotecnología Acuícola, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Viña del Mar, ChileCorrespondence A J Yáñez, R Avendaño-Herrera and J G Carcamo, Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción, Chile (e-mails: ayanez@uach.cl, gcarcamo@uach.cl, ravendano@unab.cl) Search for more papers by this authorJ G Carcamo, Corresponding Author J G Carcamo Facultad de Ciencias, Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, Chile Interdisciplinary Center for Aquaculture Research (INCAR), Concepción, ChileCorrespondence A J Yáñez, R Avendaño-Herrera and J G Carcamo, Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción, Chile (e-mails: ayanez@uach.cl, gcarcamo@uach.cl, ravendano@unab.cl) Search for more papers by this author A J Yáñez, Corresponding Author A J Yáñez Facultad de Ciencias, Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, Chile Interdisciplinary Center for Aquaculture Research (INCAR), Concepción, ChileCorrespondence A J Yáñez, R Avendaño-Herrera and J G Carcamo, Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción, Chile (e-mails: ayanez@uach.cl, gcarcamo@uach.cl, ravendano@unab.cl) Search for more papers by this authorK Valenzuela, K Valenzuela Facultad de Ciencias, Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, ChileSearch for more papers by this authorC Matzner, C Matzner Facultad de Ciencias, Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, ChileSearch for more papers by this authorV Olavarría, V Olavarría Facultad de Ciencias, Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, ChileSearch for more papers by this authorJ Figueroa, J Figueroa Facultad de Ciencias, Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, Chile Interdisciplinary Center for Aquaculture Research (INCAR), Concepción, ChileSearch for more papers by this authorR Avendaño-Herrera, Corresponding Author R Avendaño-Herrera Interdisciplinary Center for Aquaculture Research (INCAR), Concepción, Chile Laboratorio de Patología de Organismos Acuáticos y Biotecnología Acuícola, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Viña del Mar, ChileCorrespondence A J Yáñez, R Avendaño-Herrera and J G Carcamo, Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción, Chile (e-mails: ayanez@uach.cl, gcarcamo@uach.cl, ravendano@unab.cl) Search for more papers by this authorJ G Carcamo, Corresponding Author J G Carcamo Facultad de Ciencias, Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, Chile Interdisciplinary Center for Aquaculture Research (INCAR), Concepción, ChileCorrespondence A J Yáñez, R Avendaño-Herrera and J G Carcamo, Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción, Chile (e-mails: ayanez@uach.cl, gcarcamo@uach.cl, ravendano@unab.cl) Search for more papers by this author First published: 26 June 2013 https://doi.org/10.1111/jfd.12144Citations: 27Read the 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Evaluation of vaccines against P. salmonis: analysis of acquired versus innate immune response (P4055)

Abstract The Salmonid Rickettsial Septicaemia (SRS) is one of the most damaging syndrome that affects salmon farming in Chile. The causal agent of this pathology is the bacterium called, Piscirickettsia salmonis and vaccination remains the best way to reduce the economic impact caused by this disease. Several vaccines are used based in bacterins and recombinant proteins. However, the modulation of the innate and humoral immune response has not been determined. Thus 3 prototypes vaccines were used to evaluate the functionality of cellular components of P. salmonis in the modulation of immune response. In order to evaluate the immunogenicity produced by the prototype AUSTTRAL-SRS vaccine in Atlantic salmon. We designed a trial where we injected three groups of fish with three different formulations in two doses. After three weeks, the fish of each group were challenged with a lethal dose (LD70) of P. salmonis. The result obtained by studying the interleukin gene expression, ROS and lisosyme analysis let us to conclude that the three prototype vaccines were able to largely stimulate the innate immune response. However, only one prototype was efficient to stimulate the specific acquire immune systems in fish and also was highly efficient to protect the vaccinated salmon against SRS. These results indicate that the used marker of the innate immune system were not a good vaccine markers compared with acquire immune response. INNOVA 11IDL2-10523

Chemical structure of the carbohydrate backbone of the lipopolysaccharide from Piscirickettsia salmonis

Elucidation of the carbohydrate backbone structure of the lipopolysaccharide (LPS) from Piscirickettsia salmonis, the etiological agent of the salmonid rickettsial septicemia, is described. Structural information was established by a combination of monosaccharide and methylation analyses of LPS, and by NMR and mass spectrometries of oligosaccharides obtained through the use of various chemical degradations of the native polymer. The following structure of the backbone sugars was determined on the basis of the combined data from these experiments: [Display omitted] The presence of two consecutive residues of diacetylated pseudaminic acid (Pse5,7Ac, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid) in the LPS appears to be unique among polysaccharides containing this acidic sugar. Similarly, the presence of 4-aminoarabinose (Ara4N, 4-amino-4-deoxy-l-arabinopyranose) on O-4 of the α-GlcN1P of the lipid A moiety is a unique feature of this LPS.

Broth medium for the successful culture of the fish pathogen Piscirickettsia salmonis

Piscirickettsiosis or salmonid rickettsial septicaemia (SRS) caused by Piscirickettsia salmonis constitutes one of the main problems in farmed salmonid and marine fishes. Since the first reports of the disease, it has been successfully isolated and maintained in eukaryotic cell--culture systems, but these systems are time-consuming, the media are costly, and eliminating heavily contaminated host cell debris is difficult. In this report, we describe a marine-based broth supplemented with L-cysteine, named AUSTRAL-SRS broth, that facilitates superior growth of P. salmonis strains. Strains reached an optical density of approximately 1.8 when absorbance was measured at 600 nm after 6 d incubation at 18°C. Several passages (n = 6) did not alter the culture kinetics. We report for the first time the purification of DNA, lipopolysaccharide (LPS) and whole membrane protein obtained from P. salmonis grown in this liquid medium, and thus provide a suitable platform to simplify the preparation of P. salmonis cells for genetic and serological studies. Moreover, the results of the cytopathic effect test showed that P. salmonis grown in AUSTRAL-SRS broth maintained their virulence properties, inducing apoptosis after 3 d. This makes the medium a good candidate for the successful growth of P. salmonis and an excellent basis for the development of low cost vaccines.

Multiple tissue transcriptomic responses toPiscirickettsia salmonisin Atlantic salmon (Salmo salar)

The bacterium Piscirickettsia salmonis is the etiological agent of salmonid rickettsial septicemia (SRS), a severe disease that causes major economic losses to the Atlantic salmon aquaculture industry every year. Little is known about the infective strategy of P. salmonis, which is able to infect, survive within, and replicate inside salmonid macrophages as an intracellular parasite. Similarly there is little knowledge concerning the fish host's response to invasion by this pathogen. We have examined the transcriptional response of postsmolt Atlantic salmon (Salmo salar) to P. salmonis at 48 h following infection in three tissues, liver, head kidney, and muscle, using an Atlantic salmon oligonucleotide microarray (Salar_2, Agilent 4x44K). The infection led to a large alteration of transcriptional activity in all the tissues studied. In infected salmon 886, 207, and 153 transcripts were differentially expressed in liver, head kidney, and muscle, respectively. Assessment of enrichment for particular biological pathways by gene ontology analysis showed an upregulation of genes involved in oxidative and inflammatory responses in infected fish, indicative of the activation of the innate immune response. The downregulation of genes involved in the adaptive immune response, G protein signaling pathway, and apoptotic process in infected fish may be reflective of mechanisms used by P. salmonis to survive, replicate, and escape host defenses. There was also evidence of differential responses between studied tissues, with protein metabolism being decreased in muscle of infected fish and with a concomitant increase being shown in liver.

Oral vaccination of Atlantic salmon ( Salmo salar) against salmonid rickettsial septicaemia

Effective oral immunization systems may be very helpful to the salmon industry, particularly during the seawater growth stages in which vaccination through injection is not possible. During the seawater growing stage, fish become more susceptible to several types of disease, due to the natural decay of vaccine-induced immune responses. In this study, we demonstrate the immune response and efficacy of a new salmonid rickettsial septicaemia (SRS) oral vaccine, developed using MicroMatrix™ Technology. The vaccine, which is administered together with daily feed ration, induces a specific immune response at local and systemic levels. Anti- Piscirickettsia salmonis specific antibodies were detected as soon as 300 degree-days after vaccination. Furthermore, oral vaccination was able to protect fish against a lethal pathogen challenge when administered either as a primary vaccination or as a booster for an injected vaccine. Results show that oral vaccination is an efficacious treatment for the prevention of SRS outbreaks throughout the salmon culture period.

Piscirickettsia salmonis induces apoptosis in macrophages and monocyte‐like cells from rainbow trout

Piscirickettsia salmonis is the etiologic agent of the salmonid rickettsial septicemia (SRS) which causes significant losses in salmon production in Chile and other and in other regions in the southern hemisphere. As the killing of phagocytes is an important pathogenic mechanism for other bacteria to establish infections in vertebrates, we investigated whether P. salmonis kills trout macrophages by apoptosis. Apoptosis in infected macrophages was demonstrated by techniques based on morphological changes and host cell DNA fragmentation. Transmission electron microcopy showed classic apoptotic characteristics and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling showed fragmented DNA. Programmed cell death type I was further confirmed by increased binding of annexin V to externalized phosphatidylserine in infected macrophages. Moreover, significant increases of caspase 3 activation were detected in infected cells and treatment with caspase inhibitor caused a decrease in levels of apoptosis. This is the first evidence that P. salmonis induces cell death in trout macrophages. This could lead to bacterial survival and evasion of the host immune response and play an important role in the establishment of infection in the host.

Productive infection of Piscirickettsia salmonis in macrophages and monocyte‐like cells from rainbow trout, a possible survival strategy

Piscirickettsia salmonis is the etiologic agent of the salmonid rickettsial septicemia (SRS), an endemic disease which causes significant losses in salmon production. This intracellular bacterium is normally cultured in salmonid epithelial cell lines inducing characteristic cytopathic effects (CPEs). In this study we demonstrate that P. salmonis is able to infect, survive, replicate, and propagate in the macrophages/monocytes cell line RTS11 derived from rainbow trout spleen, without inducing the characteristic CPEs and the host cells showing the same expression levels as non-infected control cell. On the other hand, bacteria were capable of expressing specific proteins within infected cells. Infected macrophages cease proliferation and a fraction of them detached from the plate, transform to non-adhesive, monocyte-like cells with proliferative activity. Productive infection of P. salmonis into salmonid macrophage/monocyte cells in culture provides an excellent model for the study of host-pathogen interactions, almost unknown in the case of P. salmonis. Our results suggest that the infection of cells from the salmonid innate immune system without inducing an important cell death response should lead to the persistence of the bacteria and consequently their dissemination to other tissues, favoring the evasion of the first line of defense against pathogens.

The use of fish vaccines in the Chilean salmon industry 1999-2003

This study summarises the availability of fish vaccines in Chile in the period 1999–2003. Through a questionnaire survey, data on annual sales per product were obtained from seven pharmaceutical companies and data on the corresponding farmed salmonid population eligible for vaccination were obtained from Sernapesca. More than 20 vaccines for aquacultured fish were brought to the Chilean market during the study period. The estimated number of fish being immunized by immersion increased from 93 million to more than 200 million, whereas sales of fish vaccines for injection increased from 2,008 to 16,561 l, the latter amount corresponding to approx. 150 million doses. Estimated vaccine coverage in the susceptible species ranked from 37–84% for yersiniosis (ERM), from 51–17% for salmonid rickettsial septicaemia (SRS), and from 8–78% for infectious pancreatic necrosis (IPN). Vaccine coverage against bacterial kidney disease (BKD) remained below 5%.

Microarray analyses identify molecular biomarkers of Atlantic salmon macrophage and hematopoietic kidney response to Piscirickettsia salmonis infection.

Piscirickettsia salmonis is the intracellular bacterium that causes salmonid rickettsial septicemia, an infectious disease that kills millions of farmed fish each year. The mechanisms used by P. salmonis to survive and replicate within host cells are not known. Piscirickettsiosis causes severe necrosis of hematopoietic kidney. Microarray-based experiments with QPCR validation were used to identify Atlantic salmon macrophage and hematopoietic kidney genes differentially transcribed in response to P. salmonis infection. Infections were confirmed by microscopy and RT-PCR with pathogen-specific primers. In infected salmon macrophages, 71 different transcripts were upregulated and 31 different transcripts were downregulated. In infected hematopoietic kidney, 30 different transcripts were upregulated and 39 different transcripts were downregulated. Ten antioxidant genes, including glutathione S-transferase, glutathione reductase, glutathione peroxidase, and cytochrome b558 alpha- and beta-subunits, were upregulated in infected macrophages but not in infected hematopoietic kidney. Changes in redox status of infected macrophages may allow these cells to tolerate P. salmonis infection, raising the possibility that treatment with antioxidants may reduce hematopoietic tissue damage caused by this rickettsial infection. The downregulation of transcripts involved in adaptive immune responses (e.g., T cell receptor alpha-chain and C-C chemokine receptor 7) in infected hematopoietic kidney but not in infected macrophages may contribute to infection-induced kidney tissue damage. Molecular biomarkers of P. salmonis infection, characterized by immune-relevant functional annotations and high fold differences in expression between infected and noninfected samples, may aid in the development of anti-piscirickettsial vaccines and therapeutics.

TRNA genes were found in Piscirickettsia salmonis 16S–23S rDNA spacer region (ITS)

Piscirickettsia salmonis is the etiological agent of Salmonid Rickettsial Septicemia, a disease affecting salmon aquaculture industry. We analyzed the 16S-23S rDNA spacer region (internal transcribed spacer, ITS) of Chilean P. salmonis isolates LF-89 and EM-90. Two main ITS amplification products were obtained by PCR using L1 and G1 primers, differing from that described where only one ITS region was found. By Southern blot, it was established that these two amplification products contained sequences related to P. salmonis ITS. Sequence analysis confirmed that P. salmonis had two ITS regions: ITS A and ITS B. In both isolates, the smaller (ITS B) corresponded to ITS sequences previously described for each one, and the larger (ITS A) were almost the same as their respective ITS B sequences interrupted by an insert which contained two tRNAs genes: tRNA-Ile and tRNA-Ala.

TRNA genes were found in Piscirickettsia salmonis 16S–23S rDNA spacer region (ITS)

Piscirickettsia salmonis is the etiological agent of Salmonid Rickettsial Septicemia, a disease affecting salmon aquaculture industry. We analyzed the 16S–23S rDNA spacer region (internal transcribed spacer, ITS) of Chilean P. salmonis isolates LF-89 and EM-90. Two main ITS amplification products were obtained by PCR using L1 and G1 primers, differing from that described where only one ITS region was found. By Southern blot, it was established that these two amplification products contained sequences related to P. salmonis ITS. Sequence analysis confirmed that P. salmonis had two ITS regions: ITS A and ITS B. In both isolates, the smaller (ITS B) corresponded to ITS sequences previously described for each one, and the larger (ITS A) were almost the same as their respective ITS B sequences interrupted by an insert which contained two tRNAs genes: tRNA-Ile and tRNA-Ala.

An efficacious recombinant subunit vaccine against the salmonid rickettsial pathogen Piscirickettsia salmonis

Piscirickettsia salmonis is the aetiological agent of salmonid rickettsial septicaemia, an economically devastating rickettsial disease of farmed salmonids. Infected salmonids respond poorly to antibiotic treatment and no effective vaccine is available for the control of P. salmonis. Bacterin preparations of P. salmonis were found to elicit a dose-dependent response in coho salmon ( Oncorhynchus kisutch), which varied from inadequate protection to exacerbation of the disease. However, an outer surface lipoprotein of P. salmonis, OspA, recombinantly produced in Escherichia coli elicited a high level of protection in vaccinated coho salmon with a relative percent survival as high as 59% for this single antigen. In an effort to further improve the efficacy of the OspA recombinant vaccine, T cell epitopes (TCE's) from tetanus toxin and measles virus fusion protein, that are universally immunogenic in mammalian immune systems, were incorporated tandemly into an OspA fusion protein. Addition of these TCE's dramatically enhanced the efficacy of the OspA vaccine, reflected by a three-fold increase in vaccine efficacy. These results represent a highly effective monovalent recombinant subunit vaccine for a rickettsia-like pathogen, P. salmonis, and for the first time demonstrate the immunostimulatory effect of mammalian TCE's in the salmonid immune model. These results may also be particularly pertinent to salmonid aquaculture in which the various subspecies are outbred and of heterologous haplotypes.

Pathogenesis of liver lesions caused by experimental infection with Piscirickettsia salmonis in juvenile Atlantic salmon, Salmo salar L.

Piscirickettsia salmonis, the etiologic agent of salmonid rickettsial septicemia (SRS), or piscirickettsiosis, causes substantial economic losses to the salmon industry. The pathogenesis of the disease has not been fully characterized. The aim of this study is to describe the hepatic lesions associated with experimental P. salmonis infection in Atlantic salmon juveniles. Fish were maintained in fresh water and inoculated intraperitoneally (IP), orally, or on the gill surface with P. salmonis. A group of uninfected fish was kept as control. Liver samples from 5 fish in each inoculated group and 3 controls were collected weekly and processed for histological and immunohistochemical examination. Thickening of the liver capsule by inflammatory cells was a characteristic histologic feature of IP inoculated fish. Three weeks post-IP inoculation, 8 fish had died and 2 fish were sampled. Histological changes at this time consisted of vasculitis, presence of fibrin thrombi, vacuolated hepatocytes and focal areas of necrosis. Leukocytes containing intracytoplasmic basophilic microorganisms were seen within hepatic sinusoids. Vasculitis and intracytoplasmic vacuoles were prominent features in fish inoculated orally and on the gill surface. The presence of P. salmonis within hepatocellular vacuoles, endothelial cells, and leucocytes was confirmed by immunohistochemistry. The intracellular location of P. salmonis and the vascular damage seen in infected fish are characteristic of rickettsial infections. Histological lesions induced by experimental infection with P. salmonis using the oral and gill surface routes were similar to those observed in natural outbreaks of piscirickettsiosis. The tropism of P. salmonis for endothelial cells explains the vascular lesions observed in SRS, whereas hepatic lesions are due to ischemic necrosis and direct injury by intracytoplasmic organisms.

Purification of Piscirickettsia salmonis and partial characterization of antigens.

Piscirickettsia salmonis is the etiological agent of salmonid rickettsial septicemia, an economically significant disease affecting the salmon aquaculture industry. As with other rickettsial pathogens, antigenic analysis of P. salmonis has been limited by the inherent difficulties of purifying an intracellular organism away from host cell material. In this report, we describe the use of diatrizoate meglumine and diatrizoate sodium (DMDS) density gradient centrifugation to purify P. salmonis grown in chinook salmonis was consistently concentrated in a visible band within the DMDS density gradient at density of 1.15 to 1.16 g ml(-1). Recovery of purified, viable organisms from DMDS density gradients varied from 0.6 to 3%. Preparations of uninfected CHSE-214 cells, CHSE-214 cells infected with P. salmonis, and gradient-purified P. salmonis were compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis to assess the degree of purification and to identify P. salmonis-specific proteins. Although gradient-purified P. salmonis preparations were not completely free of host cell material, 8 bacterial proteins were identified. Polyclonal rabbit antiserum was used in an immunoblot of proteins from purified P. salmonis to identify 3 major and 5 minor antigens. The major antigens of 56, 30 and 20 kDa were potential candidates for experimental vaccines and development of novel diagnostic assays.

Salmonid rickettsial septicemia caused by Piscirickettsia salmonis:a review

Rickettsial diseases affecting several fish species have emerged in the last years. The most important outbreaks have been reported among farmed salmonid and non-salmonid species. The most important economical impact on the Chilean fish farming industry is probably the rickettsial pathogen Piscirickettsia salmonis, which is the microorganism responsible of the disease salmonid rickettsial septicemia (SRS). In the present article the authors review current knowledge on the natural history, lesions, transmission, diagnosis and control of SRS.

Antigenic characterization of the salmonid pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis, the etiological agent of salmonid rickettsial septicemia, was purified from infected immortal chinook salmon (Oncorhynchus tshawytscha) embryo cells by a combination of differential and Percoll density gradient centrifugation. Immune sera from rabbits immunized with purified whole cells of P. salmonis reacted with four protein antigens and two carbohydrate antigens with relative molecular sizes of 65, 60, 54, 51, 16, and approximately 11 kDa, respectively. The carbohydrate antigens appear to be mainly core region lipo-oligosaccharide with lesser amounts of lipopolysaccharide. Serum from convalescent rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) reacted with several minor immunoreactive protein antigens between 10 and 70 kDa in size and a carbohydrate antigen with a relative molecular size of approximately 11 kDa. The salmonid immune system did not appear to elicit a strong humoral response against this intracellular pathogen. Indirect immunofluorescence microscopy, immunogold transmission electron microscopy, and biotin labeling of intact P. salmonis cells suggest that the immunoreactive antigens identified with rabbit antisera are surface exposed and differ significantly from those identified with salmonid antisera.