microorganisms Brief Report The Analysis of Live-Attenuated Piscirickettsia salmonis Vaccine Reveals the Short-Term Upregulation of Innate and Adaptive Immune Genes in Atlantic Salmon (Salmo salar): An In Situ Open-Sea Cages Study Deborah Vargas 1,2,† , Eva Vallejos-Vidal 3,† , Sebastián Reyes-Cerpa 4,5 , Aarón Oyarzún-Arrau 1 , Claudio Acuña-Castillo 2,3 , Mónica Imarai 1,2,3 , Felipe E. Reyes-López 1,6,7, * and Ana María Sandino 1,2,3, * 1 Consorcio Tecnológico de Sanidad Acuícola, Ictio Biotechnologies S.A., 7500652 Santiago, Chile; deborah.vargas@usach.cl (D.V.); aaron.oyarzuna@usach.cl (A.O.-A.); monica.imarai@usach.cl (M.I.) 2 Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, 9170002 Santiago, Chile; claudio.acuna@usach.cl 3 Centro de Biotecnología Acuícola, Facultad de Química y Biología, Universidad de Santiago de Chile, 9170002 Santiago, Chile; eva.vallejosv@usach.cl 4 Centro de Genómica y Bioinformática, Facultad de Ciencias, Universidad Mayor, 8580745 Santiago, Chile;  sebastian.reyes@umayor.cl  5 Escuela de Biotecnología, Facultad de Ciencias, Universidad Mayor, 8580745 Santiago, Chile 6 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Citation: Vargas, D.; Vallejos-Vidal, 08193 Bellaterra, Spain E.; Reyes-Cerpa, S.; Oyarzún-Arrau, 7 Facultad de Medicina Veterinaria y Agronomía, Universidad de Las Américas, 7500975 Providencia, Chile A.; Acuña-Castillo, C.; Imarai, M.; * Correspondence: Felipe.Reyes@uab.cat (F.E.R.-L.); ana.sandino@usach.cl (A.M.S.) Reyes-López, F.E.; Sandino, A.M. The † These authors contributed equally to this study. Analysis of Live-Attenuated Piscirickettsia salmonis Vaccine Reveals Abstract: Piscirickettsia salmonis, the etiological agent of the Salmon Rickettsial Septicemia (SRS), is the Short-Term Upregulation of one the most serious health problems for the Chilean salmon industry. Typical antimicrobial strategies Innate and Adaptive Immune Genes used against P. salmonis include antibiotics and vaccines, but these applications have largely failed. in Atlantic Salmon (Salmo salar): An A few years ago, the first attenuated-live vaccine against SRS (ALPHA JECT LiVac® SRS vaccine) In Situ Open-Sea Cages Study. was released to the market. However, there is no data about the agents involved in the activation Microorganisms 2021, 9, 703. https://doi.org/10.3390/ of the immune response induced under field conditions. Therefore, in this study we evaluated the microorganisms9040703 expression profile of a set of gene markers related to innate and adaptive immunity in the context of a cellular response in Atlantic salmon (Salmo salar) reared under productive farm conditions and Academic Editor: Miguel Balado immunized with a live-attenuated vaccine against P. salmonis. We analyzed the expression at zero, 5-, 15- and 45-days post-vaccination (dpv). Our results reveal that the administration of the attenuated Received: 26 February 2021 live SRS LiVac vaccine induces a short-term upregulation of the cellular-mediated immune response Accepted: 25 March 2021 at 5 dpv modulated by the upregulation of ifnα, ifnγ, and the cd4 and cd8α T cell surface markers. Published: 29 March 2021 In addition, we also registered the upregulation of il-10 and tgfβ. Altogether, the results suggest that a balanced activation of the immune response took place only at early times post-vaccination Publisher’s Note: MDPI stays neutral (5 dpv). The scope of this short-term upregulation of the cellular-mediated immune response against with regard to jurisdictional claims in a natural outbreak in fish subjected to productive farm conditions deserves further research. published maps and institutional affil- iations. Keywords: Piscirickettsia salmonis; Salmonid Rickettsial Septicemia (SRS); attenuated live vaccine; cellular immunity; interferon-mediated immune response; open sea cage farming Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article distributed under the terms and In 2018, about 88% of the 179 million tons of total fish production was utilized for conditions of the Creative Commons direct human consumption, a share that has increased significantly in recent decades. Attribution (CC BY) license (https:// The contribution of world aquaculture to world fish production has constantly increased, creativecommons.org/licenses/by/ reaching 46.0% in 2016–2018, valued at USD 250 billion. China is by far the main world 4.0/). exporter of fish and fish products, followed by Norway, Vietnam, India and Chile. In Microorganisms 2021, 9, 703. https://doi.org/10.3390/microorganisms9040703 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 703 2 of 12 Chile, aquaculture production of Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch) and rainbow trout (Oncorhynchus mykiss) has grown strongly, thus consolidating its position as second worldwide salmon producer just behind Norway. Chile has seen sustained export revenue growth on the back of strong global demand for salmonids throughout the Americas, Europe and Asia and an increase in prices, reaching export revenues of USD 6.6 billion [1]. Under intensive culture conditions, aquatic animal disease is one of the most serious constraints to the expansion and development of sustainable aquaculture [2]. Fish may be exposed to several environmental and husbandry related stimuli that may have a potential noxious or stressful effect. All these factors have negative impacts on fish welfare, overall performance, and protective immune response, increasing the susceptibility to disease [3]. Farmed fish are continually exposed to pathogens, including bacteria, viruses, and par- asites, which may produce outbreaks and mortality [4,5]. Prevention of diseases is an advisable practice in aquaculture, which is based on improved husbandry practices, move- ment restrictions, genetically resistant-disease stock, dietary supplements, non-specific immunostimulants, vaccine, probiotics, prebiotics, medicinal plant products, water dis- infection and antimicrobial compounds as the best approaches in controlling infectious diseases of fish [4,6]. However, effective vaccines are probably the most important factors for the growth and success of intensive salmonid farming systems. The development of a sustainable aquaculture industry depends on the development and implementation of vac- cines and vaccination regimes that makes the disease situation predictable and manageable under intensive production [7]. In Chile, the National Fisheries Service (SERNAPESCA, Servicio Nacional de Pesca) identified along their coasts the presence of Piscirickettsia salmonis, the etiological agent of Salmonid Rickettsial Septicaemia (SRS). P. salmonis is probably the most serious health prob- lem for the Chilean salmon industry [8], because of its highly aggressive nature, recurrent outbreaks, and widespread transmission among other cultivated salmonid species [9–12]. In 2018, mortalities associated with P. salmonis represented 54.7% and 83.3% of the total mor- talities attributed to infectious causes in Atlantic salmon and rainbow trout, respectively [9]. The control and prophylactic strategies against P. salmonis have relied on antibiotics and vaccines to date; however, both are inadequate [13]. Antibiotics have been used inap- propriately to control outbreaks of infection, an excessive use of mainly florfenicol and oxytetracycline generating concern about public health [14]. Moreover, infected salmonids respond poorly to these treatments, likely because of the intracellular characteristics of the infective cycle of P. salmonis and the insufficient concentrations of antibiotics that reach the intracellular niche to eliminate the bacterium [13]. This situation is further complicated by the lack of effective vaccines against P. salmonis because prophylactic vaccines do not provide acceptable levels of protection [4,13]. Today, several commercial vaccines are available against P. salmonis. Most of them are based on bacterin, defined as a preparation of killed bacteria based on heat-inactivation or formalin treatment. Also available are multivalent bacterin, which contains antigens from V. ordalii (anguillarum), A. salmonicida, IPNV, ISAV and/or C. rogercresseyi. Moreover, there vaccines are available with recombinant proteins, such as AQUAVAC® SARISTIN 2, which contain VP2 protein from IPNV and ORF1-90 kDa from P. salmonis, and BIRNAGEN FORTE 2, which contains recombinant proteins HSP70, HP60 and FLG G2 from P. salmonis and inactivated IPNV. Finally, ALPHA JECT LiVac® SRS is the available live-attenuated vaccine currently in use in the Chilean salmonid aquaculture industry [13,15]. Protection induced by vaccines against P. salmonis administered under controlled conditions in the laboratory is well documented. However, protection under field con- ditions appears to be contradictory, with a lack of information regarding immune re- sponse [13,16,17]. The experience is that vaccines against P. salmonis confer good short-term protection against disease and mortality, but are inefficient in conferring long-term protec- tion [17]. Microorganisms 2021, 9, 703 3 of 12 The immune system in teleost fish has similarities to the mammalian immune sys- tem [18,19]. The innate immunity of fish is composed of physical, humoral and cellular defense barriers. The physical barriers act as the first line of defense (skin, gills and mucous membranes). The innate humoral immunity emerges from the enzymes, complement proteins and opsonizing antibodies. Finally, the cellular barrier is composed by leukocytes (macrophages, neutrophils, natural killer (NK) and eosinophils) with morphological and functional similarities to mammals’ leukocytes [19]. Nevertheless, an adaptive immunity has also been described in teleost, made of T and B lymphocytes, together with a cytokine network and a production of specific antibodies. However, the adaptive immune response has been described as slower than that of mammals, with a more limited repertoire, where specific antibodies are not detected until three or four weeks after immunization, thus making it a less efficient response than the one described for mammals [13,20]. Vaccination strategies using injectable and oral vaccines have been shown to induce an immune response mediated by antibodies [21]. However, the intracellular nature of the infective cycle of P. salmonis [13] suggests that the activation of a cellular-mediated mechanism should take place for a successful host protection. Importantly, there are no data about the mechanisms of induction for conferring the short-term protection observed in fish farming. For this reason, in this study we explored the gene expression profile of molecules associated with the cellular-mediated immune response in Atlantic salmon immunized with a live-attenuated vaccine against P. salmonis, evaluated at zero, 5-, 15- and 45-days post-vaccination (dpv). The analysis included the expression profile of innate (complement component 3 (c3); interferon alpha (ifnα)) and adaptive immune response gene markers including ifn gamma (ifnγ) (associated to T helper 1 (Th1) cells response), il-10 and tgf-β (for regulatory response), and cd4 and cd8α (T cell surface markers). We focus on the differential expression pattern between vaccinated and non-vaccinated fish. 2. Materials and Methods 2.1. Fish The study included a total of 163,590 Atlantic salmon (Salmo salar) (body weight (BW) mean 904.8 g) reared on the Chilean southern coast (Hornopirén; Región de Los Lagos). The assay was conducted in a commercial aquaculture farm under an open-sea cages productive regime. The experiment lasted for 45 days from October (11.4 ◦ C) to December 2017 (12.8 ◦ C). Water parameters were monitored daily according to the Chilean sanitary regulations in force. 2.2. Vaccination Trial and Sampling At the moment of performing the experiment, fish were already distributed in their cages for commercial purposes following routine productive practices. The trial included two sea cages: one cage for the vaccinated group (n = 80,189 fish; BW mean 1045 g; density = 4.4 Kg/m3 ); and a different cage for the non-vaccinated fish (n = 83,401 fish; BW mean 764.5 g; density = 3.4 Kg/m3 ). All non-vaccinated and vaccinated fish included in the study were weighed at zero (1143 ± 286 g) and at 5 dpv (962 ± 153 g; 1110 ± 192 g), 15 dpv (937 ± 126 g; 1167 ± 272 g), and 45 dpv (1443 ± 323 g; 1651 ± 341 g), respec- tively. Fish were starved 24 h before vaccination. The ALPHA JECT LiVac® SRS (AL 20,542 strain, 1.9 × 105 –4.9 × 106 TCID50; PharmaQ AS, Overhalla, Norway) was used as live-attenuated Piscirickettsia salmonis vaccine source. Fish were fasted for 24 h prior to vaccination. The intraperitoneal vaccination trial (100 µL per fish) was conducted after sedation with MS222 (50 mg/L) and following the manufacturer’s recommendations. In this study, the gene expression profile was evaluated in the head kidney because of the evi- dence of P. salmonis infection, survival and replication in head kidney macrophages [22,23]. Head kidney was sampled from both vaccinated and non-vaccinated fish at zero, 5-, 15-, and 45-days post-vaccination (dpv) (n = 15 fish per treatment and sampling time-point). The cumulative survival was monitored daily (99.91% survival for vaccinated fish: 99.47% for non-vaccinated fish). Before sampling, fish were euthanized by over anesthetization Microorganisms 2021, 9, 703 4 of 12 in MS222 (200 mg/L). Samples were preserved in RNAlater (Invitrogen, Thermo Fisher Scientific, Vantaa, Finland), incubated overnight (4 ◦ C), and stored immediately afterwards at −80 ◦ C until sample processing for total RNA extraction. 2.3. RNA Extraction and cDNA Synthesis Total RNA from head kidney samples (n = 15 fish per treatment and sampling time- point) was obtained using TRIsure (Bioline, London, UK). The RNA pellet was resuspended in 100 µL nuclease-free water. The total RNA was quantified spectrophotometrically with the Infinitive 200Pro (TECAN Austria GmbH, Grödig, Austria). The RNA integrity was also inspected by 1% agarose gel. Only those samples with 260:280 ratio greater than 1.7 and integrity with no apparent sign of degradation were considered. Total RNA (2.0 µg) samples were treated with the RQ1 RNase-free DNase (Promega, Madison, WI, USA) before performing cDNA synthesis using reverse transcriptase M-MLV (Promega) and Oligo dT (Promega) following the manufacturer’s recommendations. Once reactions were completed, the cDNA samples were stored until use at −20 ◦ C. 2.4. Gene Expression Analysis The head kidney from vaccinated and non-vaccinated fish were analyzed by RT-qPCR. For this purpose, the ImmuneTrack kit (Ictio Biotechnologies SA, Santiago, Chile) was used for assessing the innate and adaptive immune genes according to the manufacturer’s instructions. Thus, the expression profile of innate (c3; ifnα) and adaptive immune response gene markers including ifnγ, il-10, tgf-β, cd4, and cd8α was evaluated. We tested the expression of β-Actin (ImmuneTrack kit) and 18S (primer sequences described in [24]) in order to elucidate the better reference gene for the study. Thus, the expression of elongation factor 1 alpha (ef1α) was included as reference gene based on the low variation between all the samples included in the analysis. Importantly, the use of ef1α was suggested as reference gene in RT-qPCR assays for studying the effect of P. salmonis on the host immune response [25]. Quantitative PCR reactions were performed with 5.0 µL 2X buffer reaction SensiFAST SYBR No-ROX Kit (Bioline), 0.25 µL forward and reverse primers (10 µM concentration stock each), 3.5 µL of miliQ H2 O, and 1.0 µL from each of all the cDNA stock samples. The thermal conditions are detailed on Table 1. At the end of the last qPCR reaction cycle, a temperature ramping step from 65 to 95 ◦ C was included to produce the melting curves in order to verify the amplification of a unique single product for all the samples included in the gene expression analysis. All the reactions were performed in duplicate using the Thermo Scientific PikoReal Real-Time PCR System (Thermo Fisher, Vantaa, Finland). Quantification was done according to the Livak method [26]. The normalized relative expression (NRE) value for each condition (control and vaccinated fish) was calculated using the time zero (calibrator) and normalized to the ef1a (reference gene) expression. The results were expressed as mean expression values obtained at zero, 5, 15, and 45 days after vaccination (n = 15 fish per group, experimental condition and time-point assessed). The presence of the most infectious/persistent pathogens affecting the Chilean salmon industry including virus (IPNV, ISAV, PRV) and bacteria (P. salmonis, R. salmoninarum, and F. psychrophilum) was checked by qRT-PCR using Kit Plus (Ictio Biotechnologies SA, Chile). No amplification was registered for any of the pathogens assessed in the samples studied. Microorganisms 2021, 9, 703 5 of 12 Table 1. qRT-PCR condition details for the primers evaluated. Activation Number of Denaturation Annealing Tm Product Gene Acronym Extension Step Step Cycles Step Step (◦ C) c3 95 ◦ C × 2 min 40 95 ◦ C × 5 s 60 ◦ C × 10 s 72 ◦ C × 10 s 85.6 ifnα 40 95 ◦ C × 5 s 60 ◦ C × 10 s 72 ◦ C × 10 s 83 ifnγ 40 95 ◦ C × 5 s 60 ◦ C × 10 s 72 ◦ C × 10 s 81.8 il-10 40 95 ◦ C × 5 s 60 ◦ C × 10 s 72 ◦ C × 10 s 82.5 tgf-β 40 95 ◦ C × 5 s 60 ◦ C × 10 s 72 ◦ C × 10 s 80.1 eIFα 40 95 ◦ C × 5 s 60 ◦ C × 10 s 72 ◦ C × 10 s 85.5 18S 40 95 ◦ C × 5 s 60 ◦ C × 10 s 72 ◦ C × 10 s 82.7 cd4 95 ◦ C × 2 min 40 95 ◦ C × 5 s 57 ◦ C × 10 s 72 ◦ C × 10 s 83.8 cd8a 95 ◦ C × 2 min 40 95 ◦ C × 5 s 55 ◦ C × 10 s 72 ◦ C × 10 s 81.8 Tm: melting temperature. 2.5. Statistical Analysis All statistical analysis and graphs were conducted using Graph Pad Prism V.6.1. Software (GraphPad Software, San Diego, CA, USA). Two-way ANOVA test was used to determine differences in gene expression between control and vaccinated groups (all data were checked for normality and homoscedasticity). To find differences for each time between control and vaccinated groups, a post-hoc Sidak’s multiple comparisons test was conducted (α = 0.05). Gene expression results are expressed as the mean ±SD (standard deviation). Statistical differences were indicated when p value ≤ 0.05. To calculate the percentage of survival for control and vaccinated groups the product limit (Kaplan-Meier) method was used. 3. Results The short-term effect of the ALPHA JECT LiVac® SRS vaccine upon the modulation of immune markers was evaluated at zero, 5-, 15- and 45-days post-vaccination (dpv) in an Atlantic salmon farm located in the south of Chile and subjected to an open sea productive system. We assessed the expression of genes associated with the immune response using the ImmunoTrack I (for Innate-related genes) and the ImmunoTrack A (for Adaptive-related genes) commercial kits. Thus, the strategy included the analysis of innate (complement component 3 (c3); interferon alpha (ifnα)) and adaptive genes related to T helper (Th) response (ifn gamma (ifnγ) for Th1; interleukin (il)-10 for Th2), regulatory mechanisms (tgf-β1; il-10), and immune cell surface markers (cd4; cd8α). At innate level, we observed a slight increase in the expression of c3 at 5 dpv for the vac- cinated group (26.3 ± 16.3) compared to the non-vaccinated (control) group (11.2 ± 13.2). However, this increase in the vaccinated group did not show significant difference com- pared to the non-vaccinated treatment (p > 0.05). No differences in expression were observed at 15 and 45 dpv between the experimental groups. By contrast, ifnα showed an augmentation in its expression at 5 dpv (4.74 ± 7.75) compared to the non-vaccinated group (0.86 ± 0.94). The expression of ifnα went back to basal values at 15 (0.43 ± 0.23, respectively) and 45 dpv (0.24± 0.12, respectively) (Figure 1). These data suggest that an interferon-mediated response could take place at 5 dpv in live-attenuated P. salmonis- vaccinated Atlantic salmon. Microorganisms 2021, 9, 703 6 of 12 Microorganisms 2021, 9, x FOR PEER REVIEW 6 of 12 Figure Figure 1. 1. Normalized Normalized relative relative expression expression (NRE) (NRE) ofof immune-related immune-related genes genes for for vaccinated vaccinated (black (black dots) dots) and and non-vaccinated non-vaccinated Atlantic salmon (Salmo salar) head kidney. Fish reared under productive conditions were intraperitoneally Atlantic salmon (Salmo salar) head kidney. Fish reared under productive conditions were intraperitoneally vaccinated with vaccinated ALPHA JECT LiVac ® SRS®and sampled from both vaccinated and non-vaccinated fish at zero, 5-, 15-, and 45-days post- with ALPHA JECT LiVac SRS and sampled from both vaccinated and non-vaccinated fish at zero, 5-, 15-, and 45-days vaccination (dpv) (n = 15 fish per treatment and sampling time-point). Two-way ANOVA test and post-hoc Sidak’s mul- post-vaccination (dpv) (n = 15 fish per treatment and sampling time-point). Two-way ANOVA test and post-hoc Sidak’s tiple comparisons test was conducted (α = 0.05) to find differences in each time between control and vaccinated groups. multiple comparisons test was conducted (α = 0.05) to find differences in each time between control and vaccinated groups. Statistical differences were indicated when p value ≤ 0.05. Gene expression results are expressed as the mean ±SD. **, *** p