Why vaccines fail against Piscirickettsiosis in farmed salmon and trout and how to avoid it: A review.

Piscirickettsiosis is the most severe, persistent, and damaging disease that has affected the Chilean salmon industry since its origins in the 1980s. As a preventive strategy for this disease, different vaccines have been developed and used over the last 30 years. However, vaccinated salmon and trout frequently die in the sea cages and the use of antibiotics is still high demonstrating the low efficiency of the available vaccines. The reasons why the vaccines fail so often are still debated, but it could involve different extrinsic and intrinsic factors. Among the extrinsic factors, mainly associated with chronic stress, we can distinguish: 1) biotic including coinfection with sea lice, sealions attacks or harmful algal blooms; 2) abiotic including low oxygen or high temperature; and 3) farm-management factors including overcrowding or chemical delousing treatments. Among the intrinsic factors, we can distinguish: 1) fish-related factors including host's genetic variability (species, population and individual), sex or age; 2) pathogen-related factors including their variability and ability to evade host immune responses; and 3) vaccine-related factors including low immunogenicity and poor matches with the circulating pathogen strain. Based on the available evidence, in order to improve the development and the efficacy of vaccines against P. salmonis we recommend: a) Do not perform efficacy evaluations by intraperitoneal injection of pathogens because they generate an artificial protective immune response, instead cohabitation or immersion challenges must be used; b) Evaluate the diversity of pathogen strains in the field and ensure a good antigenic match with the vaccines; c) Investigate whether host genetic diversity could be improved, e.g. through selection, in favor of better and longer responses to vaccination; d) To reduce the stressful effects at the cage level, controlling the co-infection of pathogens and avoiding fish overcrowding. To date, we do not know the immunological mechanisms by which the vaccines against P. salmonis may or may not generate protection. More studies are required to identify what type of response, cellular or molecular, is required to develop effective vaccines.

Effectiveness of a proteoliposome-based vaccine against salmonid rickettsial septicaemia in Oncorhynchus mykiss.

Salmonid rickettsial septicaemia (SRS) is a contagious disease caused by Piscirickettsia salmonis, an intracellular bacterium. SRS causes an estimated economic loss of $700 million USD to the Chilean industry annually. Vaccination and antibiotic therapy are the primary prophylactic and control measures used against SRS. Unfortunately, commercially available SRS vaccines have not been shown to have a significant effect on reducing mortality. Most vaccines contain whole inactivated bacteria which results in decreased efficacy due to the limited ability of the vaccine to evoke a cellular mediated immune response that can eliminate the pathogen or infected cells. In addition, SRS vaccine efficacy has been evaluated primarily with Salmo salar (Atlantic salmon). Vaccine studies using Oncorhynchus mykiss (rainbow trout) are scarce, despite SRS being the leading cause of infectious death for this species. In this study, we evaluate an injectable vaccine based on P. salmonis proteoliposome; describing the vaccine security profile, capacity to induce specific anti-P. salmonis IgM and gene expression of immune markers related to T CD8 cell-mediated immunity. Efficacy was determined by experimental challenge with P. salmonis intraperitoneally. Our findings indicate that a P. salmonis proteoliposome-based vaccine is able to protect O. mykiss against challenge with a P. salmonis Chilean isolate and causes a specific antibody response. The transcriptional profile suggests that the vaccine is capable of inducing cellular immunity. This study provides new insights into O. mykiss protection and the immune response induced by a P. salmonis proteoliposome-based vaccine.

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.

Pharmacological iron-chelation as an assisted nutritional immunity strategy against Piscirickettsia salmonis infection.

Salmonid Rickettsial Septicaemia (SRS), caused by Piscirickettsia salmonis, is a severe bacterial disease in the Chilean salmon farming industry. Vaccines and antibiotics are the current strategies to fight SRS; however, the high frequency of new epizootic events confirms the need to develop new strategies to combat this disease. An innovative opportunity is perturbing the host pathways used by the microorganisms to replicate inside host cells through host-directed antimicrobial drugs (HDAD). Iron is a critical nutrient for P. salmonis infection; hence, the use of iron-chelators becomes an excellent alternative to be used as HDAD. The aim of this work was to use the iron chelator Deferiprone (DFP) as HDAD to treat SRS. Here, we describe the protective effect of the iron chelator DFP over P. salmonis infections at non-antibiotic concentrations, in bacterial challenges both in vitro and in vivo. At the cellular level, our results indicate that DFP reduced the intracellular iron content by 33.1% and P. salmonis relative load during bacterial infections by 78%. These findings were recapitulated in fish, where DFP reduced the mortality of rainbow trout challenged with P. salmonis in 34.9% compared to the non-treated group. This is the first report of the protective capacity of an iron chelator against infection in fish, becoming a potential effective host-directed therapy for SRS and other animals against ferrophilic pathogens.

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.

Gene expression associated with immune response in Atlantic salmon head-kidney vaccinated with inactivated whole-cell bacterin of Piscirickettsia salmonis and pathogenic isolates.

Piscirickettsiosis is the most challenging disease present in the Chilean salmon industry. The aim of this study was to describe the expression of genes associated with immune response of Atlantic salmon intraperitoneally infected with LF-89 and EM-90 Piscirickettsia salmonis and vaccinated with inactivated whole-cell bacterin of P. salmonis. The fish infected with PS-LF-89 showed an anti-inflammatory response, whereas this finding was not observed in the PS-EM-90-infected fish and vaccinated fish. Fish infected with both P. salmonis isolates showed mhc1-mhc2, cd4-cd8b and igm overexpression, suggesting that P. salmonis promotes a T CD4+ and T CD8+ cell response and a humoral immune response. The vaccinated-fish exhibited mhc1, mhc2 and cd4 overexpression but a significant downregulation of cd8b and igm, suggesting that the vaccine supported the CD4+ T-cell response but did not induce an immune response mediated by CD8+ T cells or a humoral response. In conclusion, the expression pattern of genes related to the humoral and cell-mediated adaptive immune response showed upregulation in fish infected with P. salmonis and down-regulation in vaccinated fish. The results of this study contribute to our understanding of the immune response against P. salmonis and can be used in the optimization of SRS prevention and control measures.

The Use of Chitosan-Coated Membrane Vesicles for Immunization Against Salmonid Rickettsial Septicemia in an Adult Zebrafish Model.

The introduction of fish vaccination has had a tremendous impact on the aquaculture industry by providing an important measurement in regard to disease control. Infectious diseases caused by intracellular pathogens do, however, remain an unsolved problem for the industry. This is in many cases directly connected to the inability of vaccines to evoke a cellular immunity needed for long-term protection. Thus, there is a need for new and improved vaccines and adjuvants able to induce a strong humoral and cellular immune response. We have previously shown that membrane vesicles (MVs) from the intracellular fish pathogen Piscirickettsia salmonis are able to induce a protective response in adult zebrafish, but the incorporation of an adjuvant has not been evaluated. In this study, we report the use of chitosan as an adjuvant in combination with the P. salmonis-derived MVs for improved immunization against P. salmonis. Both free chitosan and chitosan-coated MVs (cMVs) were injected into adult zebrafish and their efficacy evaluated. The cMVs provided a significant protection (p < 0.05), while a small but nonsignificant reduction in mortalities was registered for fish injected with free chitosan. Both free chitosan and the cMVs were shown to induce an increased immune gene expression of CD 4, CD 8, MHC I, Mpeg1.1, TNFα, IL-1ß, IL-10, and IL-6, but to a higher degree in the cMV group. Taken together, the results indicate a potential use of chitosan-coated MVs for vaccination, and that zebrafish is a promising model for aquaculture-relevant studies.

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.

Vaccines for piscirickettsiosis (salmonid rickettsial septicaemia, SRS): the Chile perspective.

INTRODUCTION: Piscirickettsia salmonis (P. salmonis) is the aetiological bacterium of the contagious disease piscirickettsiosis or salmonid rickettsial septicaemia (SRS) and causes significant economic losses to aquaculture production in Chile. Current strategies to control infection are i) indiscriminate antibiotic use and ii) vaccination with predominantly P. salmonis bacterin vaccines that do not provide acceptable levels of protection against piscirickettsiosis. Areas covered: This review covers the basic biology of P. salmonis, clinical piscirickettsiosis and disease control, the development of current P. salmonis vaccines, innate and adaptive immunity and a 5-year plan to develop new piscirickettsiosis vaccines. Expert commentary: Fundamental knowledge is lacking on the complexities of P. salmonis-host interactions, relating to bacterial virulence and host innate and adaptive immune responses, which needs to be addressed. The development of new P. salmonis vaccines needs the application of comprehensive 'omics' technologies to identify candidate vaccine antigens capable of stimulating long-lasting protective immune responses.

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.

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.

A vaccine against the salmonid pathogen Piscirickettsia salmonis based on recombinant proteins.

We report here the protective effect against piscirickettsiosis elicited in fish by a mixture of recombinant proteins. A comparative genomics strategy was used on a genomic library of Piscirickettsia salmonis in order to select optimal candidates for a recombinant subunit vaccine to protect fish from rickettsial septicaemia (SRS). Based on this information, 15 P. salmonis ORFs encoding heat shock proteins, virulence factors, membrane bound and other surface exposed antigens, were isolated and expressed. Seven of the most promising antigens were formulated in three mixtures (V1-V3) containing two or three recombinant proteins each and injected into salmon to test their protective efficacy. Two of the three formulations (V1, V2) elicited a strong protective response in a challenge against the pathogen, which was coincident with the humoral response against the corresponding recombinant proteins present in each formulation. V1, formulated with recombinant chaperonines Hsp60, Hsp70 and flagellar protein FlgG of P. salmonis achieved the highest level of protection with a relative percent survival (RPS) of 95%.

Infectivity of a Scottish isolate of Piscirickettsia salmonis for Atlantic salmon Salmo salar and immune response of salmon to this agent.

A Scottish isolate of Piscirickettsia salmonis (SCO-95A), previously shown by intraperitoneal injection to have a lethal dose (LD50) of < 2 x 10(3) infectious rickettsial units, was tested for virulence by bath challenge, surface application to the skin, or dorsal median sinus injection. Atlantic salmon Salmo salar post-smolts were used in all experiments, and exposure to 1 x 10(5) tissue culture infective doses (TCID) of P. salmonis ml(-1) for 1 h in a bath challenge resulted in only 1 mortality, 18 d later, in 10 exposed fish. Application of 2.5 x 10(6) TCID of P. salmonis SCO-95A to paper discs on the skin failed to induce any mortalities within 42 d. Intraperitoneally, fish were administered vaccines containing 10(9) heat-inactivated (100 degrees C, 30 min) or 10(9) formalin-inactivated P. salmonis SCO-95A in adjuvant, with a control group receiving phosphate-buffered saline (PBS) in adjuvant. After an induction period of over 6 mo fish were challenged by injection of P. salmonis into the dorsal median sinus. Mortalities in the control group reached 81.8% and the heat-inactivated and formalin-inactivated vaccines gave significant protection from P. salmonis, with relative percentage survivals of 70.7 and 49.6%, respectively. The nature of the protective antigen is unknown, but could be lipopolysaccharide or a heat-stable outer membrane protein. Fish that survived a dorsal median sinus challenge of P. salmonis or were cohabitants showed a strong immune response to P. salmonis.