Identification of Research Gaps for Highly Infectious Diseases in Aquaculture the Case of the Endemic Piscirickettsia Salmonis

Aquaculture 482 (2018) 211–220

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Aquaculture

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Identiﬁcation of research gaps for highly infectious diseases in aquaculture: MARK The case of the endemic Piscirickettsia salmonis in the Chilean salmon farming industry

⁎ Fernando O. Mardonesa, ,1, Felipe Paredesb,c,1, Matías Medinad, Alfredo Telloe, Victor Valdiviab, ⁎⁎ Rolando Ibarrae, Juan Correaf, Stefan Gelcichb,f, ,1 a Escuela de Medicina Veterinaria, Facultad de Ecología y Recursos Naturales, Universidad Andres Bello (UNAB), Republica 440, Santiago, Chile b Center for Applied Ecology and Sustainability (CAPES), Pontiﬁcia Universidad Católica de Chile (PUC), Santiago, Chile c Departamento de Areas Protegidas, Ministerio de Medio Ambiente, Santiago, Chile d Blue Genomics Chile, San Francisco 328, Puerto Varas, Chile e Instituto Tecnológico del Salmón (INTESAL de SalmonChile), Av. Juan Soler Manfredini 41, OF 1802 Puerto Montt, Chile f Facultad de Ciencias Biologicas, Pontiﬁcia Universidad Católica de Chile (PUC), Santiago, Chile

ARTICLE INFO ABSTRACT

Keywords: Salmonid rickettsial septicemia (SRS) or piscirickettsiosis has historically been the most important health pro- Infectious diseases blem of farmed salmonids during the growth-out production phase in the Chilean industry. SRS is caused by the Knowledge gaps bacterium Piscirickettsia salmonis and is responsible for about 50.5 to 97.2% of the total disease-specific salmon Piscirickettsia salmonis mortality in the industry. Although SRS is also prevalent in Norway and Canada, its impact on the farmed salmon Salmon farming industry of those countries are less detrimental than in Chile. Based on a comprehensive literature review and a Sustainable aquaculture participatory priority-setting workshop with key stakeholders, we show how science-based research on SRS has Science-based policy evolved over time and identify 8 main research areas which should be addressed. These areas, termed epidemiology, ecology and environmental science, microbiology, immunology, pharmacology, “Omics”, human dimensions and vaccine development include a set of 52 specific research questions to be tackled. These research areas and specific questions need to be developed based on an integrative, collaborative and crosscutting interaction in order to be successful. A long term approach based on a research center led from within government agencies, co-financed by the salmon industry should be developed in order to foresee how research gaps must be adaptively modified in order to address the impacts of P. salmonis over the productivity of the salmon farming industry, the physical and ecological environment, and the socio-economic sustainability. This approach could result in significant gains for the environment and the industry and generate novel cross-sector alliances.

1. Introduction pathogen-environment model (Jones et al., 2008; Groner et al., 2016). A change in these factors can lead towards or away from a diseased Infectious diseases in aquatic ecosystems are important drivers that state, and disease emergence is controlled by this relationship. The can adversely impact biodiversity. Marine diseases are a natural part of factors that influence disease occurrences and severity occur across ocean ecosystems, but many have economic consequences for fisheries many scales, from pathogen-antigen interactions, to trait- and density- or aquaculture (the farming of aquatic organisms) (Lafferty et al., mediated responses of hosts to pathogens and from local, to global 2015). Their study is becoming increasingly important as anthro- environmental fluctuations. Understanding how processes interact pogenic forcers that may exacerbate certain disease processes across these scales to modify epidemiological patterns is a key chal- (Beardmore et al., 1997). International, national and local-scale policies lenge, for which numerous new multidisciplinary fields are emerging, increasingly call for actions to improve ocean ecosystem health including molecular epidemiology, genomics, data mining, and science- (Halpern et al., 2015), recognizing that the emergence, magnitude, and based policy approaches (Restif et al., 2012). Moreover, the “One outcome of diseases are driven by complex interactions within the host- Health” concept has been extensively used to describe those practices

⁎ Corresponding author. ⁎⁎ Corresponding author at: Center for Applied Ecology and Sustainability (CAPES), Pontiﬁcia Universidad Católica de Chile (PUC), Santiago, Chile. E-mail addresses: [email protected] (F.O. Mardones), [email protected] (S. Gelcich). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.aquaculture.2017.09.048 Received 26 January 2017; Received in revised form 24 September 2017; Accepted 30 September 2017 Available online 02 October 2017 0044-8486/ © 2017 Elsevier B.V. All rights reserved. F.O. Mardones et al. Aquaculture 482 (2018) 211–220

About P. salmonis, About P. salmonis, Fig. 1. Graphical representation of steps and ﬁ what is unknown? what is known? processes involved in the identi cation of research gaps.

Expert consultation with Literature search and industry representatives systematic review

Cross-matching of research questions

Extraction of metrics and indicators

Identification of research gaps

that support transdisciplinary collaborations involving animal and effects on the environment and public health, causing public concerns human health and the environment (Osburn et al., 2009) and likely (Buschmann et al., 2012). should be the approach needed for aquaculture worldwide, as has been From the sanitary point of view, controlling SRS has been difficult in recognized for the management of infectious diseases in oyster aqua- Chile mainly due to the wide-spread and high degree of endemicity of culture (Pernet et al., 2016). the pathogen in the salmon farming regions (Rozas and Enriquez, Aquaculture provides about 50% of the world's supply of seafood, 2014). The probability of reporting mortalities due to SRS in any pro- with a value of US$160.2 billion (FAO, 2016). In 2013, fish accounted duction cycle has been estimated in 82.5%, suggesting that neither the for about 17% of the global population's intake of animal protein and current use of vaccines or antibiotics against P. salmonis have been ef- 6.7% of all protein consumed. Overall, fisheries and aquaculture assure fective strategies to eliminate the infection. Thus, to control and ef- the livelihoods of 10–12% of the world's population and employed an fectively reduce the effects of SRS it is important to identify knowledge estimated 24 million people (FAO, 2016). For certain countries such as gaps which can provide guidance on how to address critical questions Chile, the emergence and development of the salmon farming industry regarding ways to deal with the SRS problem (Robinson et al., 2011a). has become one of the main export sectors and a significant contributor While research on SRS has been performed during the past two decades, to regional development. After 35 years, salmon farming in Chile is the there has not been a broader scale review and synthesis of scientific most important animal production system, largely driven by economic research on SRSs which can determine critical knowledge gaps to im- gains, and the second largest salmon producer in the world. Currently, prove the performance of strategies to solve the SRS crisis. salmon farming represents 36% of food exports and provides direct and The aim of this work was to review the current status of scientific indirect employment to more than 70,000 people, many in remote knowledge of SRS, the most important disease affecting farmed salmon areas. in Chile, and to identify key research gaps needed to support fish health The global salmon sector is increasingly threatened by emerging management by reducing the impact of SRS. Moreover, these results are infectious diseases, which have caused substantial problems and costs integrated into a coherent multidisciplinary research agenda, based on for the industry (Pettersen et al., 2015). Salmon farming in Chile has specific research questions that if answered, will prove the basic faced a myriad of challenging issues including production stability, building block to confront this infectious disease. sustainability, social transformations, and disease outbreaks. Indeed, fi infectious diseases have been identi ed as the major obstacle to salmon 2. Methods farming's growth. Diseases have increased steadily since the beginning ffi fi of the industry; o cial sh health reports indicate that at least 15 Two sources of information were attained by different procedures to different infectious agents have been diagnosed from Chilean farmed get insight into the current status of the scientific knowledge regarding salmon. These can contribute from ca. 9 to 50% of the total mortality SRS, and the identification of research gaps. These sources of in- during a production cycle. Salmonid rickettsial septicemia (SRS) or formation were generated by combined methodologies that are based piscirickettsiosis has historically been the most important health pro- on eliciting expert opinion adapted to identify SRS-specific scientific blem in this industry during the growth-out production phase. gaps (Sutherland et al., 2006; Sutherland et al., 2011) and, by a lit- SRS is caused by the bacterium Piscirickettsia salmonis and is re- erature search and systematic review of available scientific literature sponsible for ca. 50 to 97% of the total disease-specific salmon mor- (Robinson et al., 2011a; Robinson et al., 2011b). A third step included a tality in the industry; accounting for annual direct and indirect loses cross matching process. Each step is described in Fig. 1. between US$ 300–500 million (Cabezas, 2006). Infected fish are ne- gatively affected by loss of appetite, reduced growth, reduced food conversion efficiency, additional stress, secondary infections, depres- 2.1. Eliciting expert opinion sion of the immune system, skin damage, and mortality. At the farm fi level, SRS is costly because of drugs, reduced marketability, mortalities To identify speci c aspects of the disease in which key stakeholders fi and more expensive farming practices. Finally, antibiotics, and any felt unaware about scienti c knowledge, we applied a participatory fi other chemical product used to control infections, might have negative priority-setting exercise for the identi cation of research questions (Sutherland et al., 2006, 2011). A preliminary face-to-face interview

212 F.O. Mardones et al. Aquaculture 482 (2018) 211–220 was carried out with key stakeholders that were selected by targeting given research question could be fully answered from the published audiences that included policy makers and practitioners, funders of literature, such question was excluded from the final list provided by research and researchers in public, private and non-profit organiza- the experts. Conversely, questions without an answer, or any failure to tions. The main goal of this exercise was to identify a set of research identify an appropriate answer, were kept in the final list and where questions that would increase the effectiveness of sanitary management referred here as a research gap. Subsequently, each of the research gaps regarding SRS. Eliciting expert opinion has been proved to be successful was assigned to a discipline and sub discipline from the OCDE, and and scientifically recognized activities where information is not avail- finally they were grouped into major topics to end up with core re- able, and similar approaches have been used for other infectious dis- search areas. The process for the identification of research gaps is eases affecting farmed salmon (Gustafson et al., 2005; Gustafson et al., summarized in a flow diagram (Fig. 1). 2013; Oidtmann et al., 2014). After the preliminary interview, the research team identified a range 2.4. Analysis of results of topics. These were used as a guide for a two-day workshop with all the experts, led by the Technical Institute for Salmon (INTESAL; authors The number of P. salmonis publications was plotted against pub- MM, AT, RI). Topics of the workshop included: (1) the general char- lication year to assess changes over time and to estimate any linear acteristics of P. salmonis and SRS (the disease), (2) immunology and trend assessed by ordinary regression techniques. Histograms were vaccines, (3) therapy and antimicrobial susceptibility, (4) nutritional constructed to evaluate the frequency distribution of published data aspects regarding fish health and SRS, (5) genetic resistance to P. sal- and results from the expert consultation. To visualize the network for monis and (6) health management. Invitations to participate in the international collaborations we used the free software R (R Core Team, workshop were sent by INTESAL and included national and interna- 2016), and the ggplot2, maptools, geosphere and png packages. tional experts. The two-day workshop was held in November 2014. During the first day, each topic was discussed and potential knowledge 3. Results gaps identified. Day two was aimed at summarizing conclusions of the main topics and also considered a final session aimed at seeking con- 3.1. Identification of research gaps from the expert workshop sensus on the main knowledge gaps. The participatory workshop included a panel of 21 experts in the 2.2. Literature search and systematic review fields of molecular biology, pharmacology, nutrition, pathology, immunology, genetics, epidemiology and salmon production. Experts Literature searches were conducted in English and Spanish to generated a consensual list with 53 research questions (supplementary identify all investigations related to the infection caused by P. salmonis information), which were grouped into 9 subjects including ecology, in wild and farmed salmonids. As in previous research (Mardones et al., epidemiology, “Omics”, immunology, microbiology, nutrition, ocea- 2010), we systematically searched scientific literature from two main nography, pharmacology and policy. Most research questions fell into electronic databases: Web of Science (https://webofknowledge.com/) the field of the epidemiology and “Omics” (22% each), followed by and PubMed (https://www.ncbi.nlm.nih.gov/pubmed/) that were ex- pharmacology (17%), and immunology (12%) (Fig. 2). plored through the local server of the Pontificia Universidad Católica de Chile (PUC) using the multiple keywords and expressions (salmon* OR 3.2. Literature review and systematic review Oncorhy* OR trout OR coho OR salar OR farmed OR wild OR aqua- cult*) AND (piscirickett* OR salmonis) AND (infect* OR transm*). The number of publications over time for P. salmonis ranged be- References cited in retrieved reports were reviewed to identify addi- tween two and eighteen articles per year, with an important increase tional reports, which, if not available on line, were requested and since 2009 (Fig. 3). Since 1990, a total of 168 peer-reviewed papers scanned through the PUC library. Titles and abstracts were imported have been published. From 1990 to 2013, there was no visualization of into a reference manager system (EndNote ®). The review was restricted a linear trend and published papers main themes changed continuously to include publications from 1 January 1985 through 1 May 2016. over time, with the visual identification of certain periods where spe- Five randomly selected publications were reviewed independently cific research groups were leading P. salmonis investigations. Al least by two authors of this paper, and each of the reviewers independently three peaks were identifiable: 1995–1997, 2002–2005, and after 2009. created a spreadsheet (Microsoft Excel ®), in which each row re- Overall, a total of 96 collaborative institutions from 18 countries presented an individual reference containing information including the had investigated P. salmonis through the last 16 years (Figs. 4 and 5). Of publication year, address of corresponding author, co-authors' names these countries, Chile (48%) and the USA (15%) have collaborated in and last names, institutions and country, a concise description of the almost half of the total published work. Other countries that have major conclusions or findings, research topic and, if reported, project collaborated importantly are Canada (8%), Scotland (6%) and Norway number and/or funding agencies. Following, criteria for inclusion or (5%) (Fig. 4). In Chile, at least 4 institutions have established strong exclusion of papers and a template for data collection were discussed collaborations overseas (Fig. 5). These institutions are the School of and agreed upon by the authors. Subsequently, a double independent Veterinary Medicine of the University of Chile (FAVET, Santiago, extraction of information, metrics and indicators from the publications Chile), Bios Chile (Santiago, Chile), the Laboratorio de Genética e In- was conducted by two of the authors. Extracted data were reviewed by munología Molecular at the Pontificia Universidad Católica de Val- four of the authors and in case of disagreement, a second revision of the paraíso (LGIM/PUCV, Valparaíso, Chile) and the Universidad Austral de manuscript was jointly conducted by the authors to reach consensus on Chile (UACh, Valdivia, Chile). In USA, most research has been done by the interpretation of the results. Findings from each publication were the Oregon State University (OSU, Oregon, USA) and the School of assigned to 1 to 3 topics or sub disciplines according to the Revised Veterinary Medicine at the University of California, Davis (UC Davis, Field of Science and Technology classification of the Organization for California, USA). Economic Cooperation and Development (OCDE, http://www.oecd. Papers were allocated to 19 research topics. Most published papers org/science/inno/). were focused on aspects of the pathogenesis of the bacterium (20.3%), case reports and the epidemiology of the infection (13%), and devel- 2.3. Identification of research gaps opment of diagnostic tests (10.6%). An important number of papers (n = 12, 9.8%) reported the identification of Rickettsia-like organisms, In this step, each research question generated after the expert eli- Piscirickettsia-like organisms, and Francisella-like bacteria, which are citation workshop was revisited along with the literature search. If a almost identical to P. salmonis, however, the relationships among these

213 F.O. Mardones et al. Aquaculture 482 (2018) 211–220

Fig. 2. Research subjects as a percentage of research gaps (n = 53) that were identified by a group of experts in a 2-day workshop in Puerto Montt, Chile. have not been fully evaluated. Results allow to identify 8 research areas that need prompt attention The greatest number of papers were published in 2015 (n = 18). and funding. We briefly discuss important research gaps associated to These show a focus on host resistance (n = 6), followed by those who each and conclude by outlining a possible strategy to confront these assess infection rates and strategies (n = 4) and immunization studies from a multidisciplinary national level standpoint. (n = 4). One of the key knowledge gaps identified in current research of P. salmonis epidemiology (1) considers the need to have estimates of sensitivity and specificity as well as the predictive values to develop 3.3. Identification of research gaps and definition of research lines new and improve current diagnostic tests. Fish health specialists make reasoned judgments or give assistance under uncertainty about a po- The cross tabulation between the expert workshop and the literature tential threat, or more specifically, the diagnosis of P. salmonis affecting review identified a total of 52 research questions (RQs) which corre- fish populations. Establishing diagnoses is an imperfect process, re- sponded to almost the same identified by the experts but one. The de- sulting in a probability rather than a certainty of being right. Arguably, finitive 52 RQs were grouped into 19 research topics (RTs), and 8 re- clinical manifestations differ slightly between and among fish diseases; search areas (RAs) (Table 1). These RAs included: (1) epidemiology, (2) many of them share common behavioral and gross external clinical ecology and environmental sciences, (3) microbiology; (4) im- signs and risk factors and result in syndromes that, in terms of pro- munology, (5) pharmacology, (6) “omics”, (7) human dimensions; and duction, are undistinguishable from each other. A diagnostic test able to (8) vaccine development. The number of RQs per RTs ranged from 1 to produce estimates of the probability that a given syndrome was asso- 5, whereas the number of RTs per RAs ranged from 1 to 3. The number ciated with a given disease, depending on the nature of the observed of RQs per RAs ranged from 3 (vaccine development) to 10 (epide- syndrome and farm risk factors would help to implement preventive miology). measures, and also to reduce significantly the risk of infection in off- spring by discarding eggs from infected parents (broodstock screening). 4. Discussion Moreover, a correct classification of disease status for a given epidemiological unit under study (e.g., fish, net pen, farm, etc.) will allow the In this study, we identified key research areas needed to enhance identification of risk (or protective) factors and their relative im- the body of knowledge of one of the most critical and costly bacterial portance for the time to infection, establishment of clinical signs and disease affecting farmed salmonids in the second largest salmon in- overall magnitude of SRS. Such heterogeneity can be captured by the dustry in the world. Considering that an average scientist for almost any design and implementation of disease spread simulation model for P. discipline in developing countries publishes about 2 to 7 scientific ar- salmonis infections that ultimately will serve as a tool for the prediction ticles per year (Fanelli and Larivière, 2016), the historical status of and evaluation of control strategies (deJong, 1995; Murray, 2013; scientific knowledge for P. salmonis can be considered as deficient, as Salama and Rabe, 2013). Most publications related to this field were the median number of publications per year is about 5 since 1990, restricted to case descriptions, mortality patterns, and some associa- which may be the equivalent to the work of 1 or 2 average scientists tions with species, temporal or spatial features. Thus, epidemiological alone. Over time, this knowledge has shown irregular patterns with studies aimed to identify risk factors for SRS and the development of predominance on the topics of pathogenesis and epidemiology. Pre- spread models that mimic the transmission processes within- and be- vious to the last assessed 8 years, two “waves” or peaks of knowledge tween-host are key knowledge gaps which must be resolved regarding are described, in which 6 institutions (4 in Chile and 2 in the USA) SRS. concentrate about 60% of the total knowledge reported for P. salmonis.

214 F.O. Mardones et al. Aquaculture 482 (2018) 211–220

Fig. 3. Timeline for the number of publications retrieved through a literature search and systematic review process. Dashed line is the median number of publications for all years (median = 5). Blue lines with brackets denotes the 18 time periods where some institutions were leading research. Note that 2016 reports are assessed only until May 1st. (For interpretation of the references to colour in this FAVET−Chile ﬁgure legend, the reader is referred to the web version of this article.) 16 UACh

PUCV

10 FAVET−Chile BIOS−Chile

8 Number of papers related to P. salmonis

1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 Year of publication

The research area named ecology and environmental sciences (2) of the bacterium (Garces et al., 1991; Fryer and Lannan, 1996; Smith stresses the need to study the effects of the ecosystem on the pathogen's et al., 1996), there is too much uncertainty about the mechanisms that dynamics. The role of vector diversity and other non-salmonid fish that may be involved with vertical transmission and the role of fresh and salt could eventually act as reservoirs for P. salmonis is unknown. In addi- water in the life stage, viability and virulence of the pathogen. On the tion, the consequences of nutrient superabundance and its role in the other hand, there is an important research gap in elucidating P. salmonis survival and persistence of the pathogen is a key research gap. survival mechanisms and how these mechanisms could differ in terms Hopefully, future studies should identify attributes of the ecosystem of host susceptibility and the bacteria's population structure (Falush that might shift it toward stabilizing negative feedback (SRS outbreaks et al., 2003). A third topic that requires further research in this research reducing the likelihood of subsequent disease), versus positive feedback line relates to the emergence and frequency of P. salmonis antibiotic (SRS outbreaks facilitating subsequent pathogen attacks). Likewise, resistant strains. Understanding of the processes and timing related to studies on the biological and non-biological characteristics of the the emergence of drug resistance would allow the identification of more marine ecosystem and geographic zones (i.e., seascape ecology) are effective drugs, schemes and strategies to be implemented to avoid drug needed to assess the influence of broader scale factors on the population resistance. Chilean farmers have been forced to increase antibiotic use density and movement patterns of agents, vectors, and transmission largely due to the P. salmonis problem, the lack of an effective vaccine, stages. Such characterization would offer new knowledge for a more and the emergence of antibiotic resistance. Any improvement could comprehensive zonification strategy for salmon farming and disease greatly reduce antibiotic use. management. Dynamics of the immune response and co-infections mechanisms The research line termed microbiology (3) has important research are the topics that require critical attention within the research area of gaps. Laboratory and field experiments able to increase the basic immunology (4). Specifically, there is little research on the defense knowledge behind the morphological, structural and physiological as- mechanisms to clear intracellular bacteria (clearance). The intracellular pects driving the success of P. salmonis are still needed. Despite many degradation process of autophagy has recently shown to have different early investigations on SRS were focused on aspects of the pathogenesis roles during different bacterial infections that restrict bacterial

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F.O. Mardones et al. Aquaculture 482 (2018) 211–220

ail

a rtsuA

Colombia 1% Peru 1% France 1% Iran 1% Ireland 1% Turkey 1% Argentina 1% Taiwan 1% UK 1%

Japan 2%

Greece 3%

Spain 3%

Norway 5% Chile 48% Scotland 6%

Canada 8%

USA 15%

Fig. 4. Pie chart with the contribution at country level of collaborative centers listed in the total of peer-reviewed publications for Piscirickettsia salmonis (n = 145).

P. salmonis research network Fig. 5. Piscirickettsia salmonis research network. A black dot indicates the location of a lead in- stitution according to the corresponding address listed from retrieved papers. Grey dots are sec- 60 ondary collaborative institutions from co-au- OSU thors.

PUCV FAVET -40 Bios Chile UACh

-150 001- -005 050001 51

216 F.O. Mardones et al. Aquaculture 482 (2018) 211–220

Table 1 The 52 research questions (RQs) identiﬁed in the study grouped by research areas (RAs) and research topics (RTs) after a process of systematic review and expert elicitation for P. salmonis.

Research area (RA) Research topic (RT) Number Research question (RQ)

I. Epidemiology A. Assessing the validity and reliability of 1 What are the sensitivity and specificity of diagnostic tests for P. salmonis detection? diagnostic and screening tests 2 Can the results obtained be replicated if the test is repeated? (this is related to the reliability or repeatability of the tests) 3 Which factors can influence validity and reliability of diagnostic test, and how to use such information for better disease diagnosis and management? B. Identification of risk factors for infection, 4 Which factors (risk or protective) can modulate the time to infection, clinical type clinical signs and magnitude and magnitude of SRS outbreaks? If any, how they act or interact? 5 Which factors are associated with salmon farms that may shed P. salmonis in a higher rate compared to others? 6 How these factors can contribute to developing optimal management strategies at the farm and neighbor levels? C. Epidemiological modeling; within- and 7 Which mechanisms influence the re-emergence of P. salmonis in a farm, and which between-host transmission biological and non-biological mechanisms participate in the transmission and further spread of the bacteria within a farm? 8 How P. salmonis spreads from an infected farm to others (between-farm spread) and neighborhoods? 9 Are within- and between-farm spread related? How these two mechanisms can be coupled in an explanatory/predictive model for the use of the industry/ government? 10 Can a disease spread model(s) be designed and implemented as a tool for an early warning system, and to evaluate management strategies or treatments schemes that ultimately support the decision-making process? II. Ecology and environmental A. The effect of ecosystem on P. salmonis 11 What is the role of vector diversity and (non) salmonid reservoirs in the P. salmonis sciences dynamics dynamics? 12 What are the consequences of nutrient superabundance on pathogens, parasites, and disease? 13 What attributes of the ecosystem might predispose it toward stabilizing negative feedback (SRS outbreaks reducing the likelihood of subsequent disease), versus positive feedback (SRS outbreaks facilitating subsequent pathogen attacks)? B. Seascape structure, disturbance, and disease 14 How the seascape structure (including oceanography and abiotic factors) influences dynamics population density and movement patterns of agents, vectors, and transmission stages? 15 How can knowledge of seascape structure be used to improve quantitative predictions about disease spread and persistence? III. Microbiology A. Biology and virulence of P. salmonis 16 Which mechanisms are involved in the vertical transmission of P. salmonis? 17 What is the role of fresh and salt water in the life stage, viability and virulence of P. salmonis? 18 How P. salmonis strains differ in terms of pathogenicity and virulence, and how the host responds to these differences and signals? B. Outside-host biology of P. salmonis 19 How P. salmonis interact with surfaces, how they survive outside of their hosts, how signals are relayed between the microorganism and the host? 20 How P. salmonis strains differ in terms of host susceptibility and geographical zones? How is the population structure? C. The emergence of antibiotic resistance 21 What is the frequency of P. salmonis resistant strains, if any, before antimicrobial use in a farm? 22 What are the processes related to the emergence of drug resistance, and timing the emergence of resistance of P. salmonis? 23 What are the best drugs, schemes and strategies to be implemented in order to avoid drug resistance? IV. Immunology A. Dynamics of immune response 24 What are the conditions that specify whether P. salmonis will establish a persistent infection or will be cleared by the fish immune response? 25 What is the correlation between bacterial dynamics, damage and inflammation to the fish, and the fish response? the correlation between bacterial load (abundance of P. salmonis) and the magnitude of the antibacterial immune response? 26 How the use of vaccines or alternative products aimed to enhance the force of the immune response will decrease fish susceptibility and decreased shedding rates? B. Co-infection mechanisms (e.g., sealice, 27 What are the mechanisms involved in the host-multipathogen responses? What is BKD) the role of stress and immunity threshold? 28 Is there any temporal succession pattern of different diseases? (continued on next page)

217 F.O. Mardones et al. Aquaculture 482 (2018) 211–220

Table 1 (continued)

Research area (RA) Research topic (RT) Number Research question (RQ)

V. Pharmacology A. Pharmacokinetics and pharmacodynamics 29 What is the most effective antibiotic currently available to treat infected salmon? How is their sensitivity? 30 How different are the pharmacokinetics and pharmacodynamics of these drugs? Which factors can modulate these processes? 31 How to measure effective plasmatic concentrations and how correlated are the antimicrobial susceptibility in vitro and in vivo with treatment success? B. New generation of drugs and alternative 32 How to ensure an optimal performance for a new antibiotic (safety, effectiveness, (non-pharmacological) treatments low cost, easy to apply, etc.)? 33 How to develop and use alternatives drugs (probiotics, additives, other immunostimulant, etc.)? How to ensure their optimal performance? C. Drugs schemes and strategies 34 How to achieve the prescribed dose in each treatment? 35 What rotation scheme of different drugs minimizes the resistance of P. salmonis to drugs? 36 Is the spatial scale of salmon farming “barrios” appropriate? Do they need to be coordinated in the control of P. salmonis? 37 What are the ecological impact and costs of these rotation schemes? VI. “Omics” A. Fish mechanisms for infection resistance 38 What are the areas of the genome (genetic markers) that encode for fish resistance mechanisms to P. salmonis? 39 Are the genetic resistance mechanisms described the same in experimental versus natural infections? How to standardize such evaluation? 40 How genetic selection for disease resistance could interfere with desirable production and health-related characteristics? B. Agent mechanisms for drug resistance 41 What are the areas of the genome (genetic markers) that encode for agent resistance mechanisms to antibacterials? 42 Are the genetic resistance mechanisms described the same in experimental versus natural infections? How to standardize such evaluation? VII. Human dimensions A. Disease management 43 How to develop an interdisciplinary research framework to optimize approaches and interventions needed to reduce disease risk to farmed salmon? B. System optimization of disease 44 How to measure the impacts and effectiveness of animal health decisions including management cost-benefit analysis, cost-effectiveness, welfare measures, externalities, risk, asymmetric information, strategic behavior, and others? C. Science-policy interphase 45 How to develop indicators for scientific-based norms? 46 What is the interplay between environmental norms and policy? 47 How much policy coherence is found in the salmon aquaculture sector? 48 How can stakeholder perceptions inform the policy-making process? 49 What are the benefits and costs of implementing such policies? VIII. Vaccine development A. Vaccine development 50 What are the most relevant agent factors that may be useful to develop an efficacious vaccine against P. salmonis? 51 How to enhance the uptake, processing and presentation of the antigen to the fish innate immune system at the mucosal level as a booster for vaccination strategies? 52 How to define a standard and adequate method to assess vaccine safety, efficacy and transmissibility? replication, act in cell autonomous signaling or support bacterial re- efficacy and cost, environmental welfare and potentially to human plication. Autophagy pathway and proteins balance the beneficial and safety (Rigos and Smith, 2015). In addition, the market entrance of new detrimental effects of immunity and inflammation (including damage), generation's drugs and alternatives (non-pharmacological) treatments and thereby may protect against SRS (Levine et al., 2011). Research require a direct evaluation in the field including fish safety and the which addresses these mechanisms would provide knowledge for new success of the prescribed dose in each treatment. There is no published products that would enhance the force for the immune response that work that developed harmonized schemes for monitoring antimicrobial ultimately will decrease fish susceptibility and, supposedly, shedding resistance and effectiveness against P. salmonis, neither the ecological rates. impact nor costs associated with treatment strategies. Consequently, In this research area, the study of the immunological mechanisms this research area should provide essential information for a judicious involved in the host-multipathogen responses were identified as im- use of antimicrobial for fish health veterinarians and the industry. portant research lines. This research is critical to provide key in- The main gap identified here refers to those studies that consider the formation about the interaction, if any, between P. salmonis and one or application of genetic and genomic techniques to disease resistance, the more from the at least 15 different infectious agents that have been interpretation of data arising from such studies and the utilization of isolated from official surveys from farmed salmon in Chile (Sernapesca, the research outcomes to breed animals for enhanced resistance. Here, 2015). Co-infections between virus, parasites and bacteria have been “Omic” technologies adopt a holistic view of the molecules that make reported in most regions where salmon farming activities occurs up a cell, tissue or organism. They are aimed primarily at the universal (Valdes-Donoso et al., 2013; Karlsen et al., 2014; Valdenegro-Vega detection of genes (genomics), mRNA (transcriptomics), proteins (pro- et al., 2015). teomics) and metabolites (metabolomics) in a specific biological sample Key aspects on the control of P. salmonis are related to the strategy in a non-targeted and non-biased manner. Genetics of P. salmonis re- of the application of mass treatment to affected fish, and type of drug sistance, including genomics, has been identified in this study as a gap used. These aspects are included into the research line of pharmacology and its development, within others, should focus on the identification of (5) that deals with a number of uncertainties regarding the effectiveness fish mechanisms for infection resistance. Since the time when the of a given treatment and scheme provided that antimicrobials are analyis presented here was performed, important research has begun to needed for the relief of pain, and suffering in fish. Thorough knowledge address these questions (Pulgar et al., 2015; Correa et al., 2017; and integration of appropriate drug pharmacokinetics and pharmaco- Tarifeño-Saldivia et al., 2017). Additionally, it is important to under- dynamics in therapeutic schedules are crucial in terms of treatment stand if genetic selection for disease resistance could interfere with

218 F.O. Mardones et al. Aquaculture 482 (2018) 211–220 desirable production and health-related characteristics (Yanez et al., 2007). It is also necessary to gain insights about the underlying me- 2014). chanisms to enhance the uptake, processing and presentation of the The interplay between environmental norms and policy develop- antigen to the fish innate immune system at the mucosal level, parti- ment, policy coherence, and science-based pathways for policy devel- cularly addressing the importance of boosters for vaccination strategies. opment are the three components of the so-called human dimension In particular, there is a lack of field information regarding the immune research area. A clear institutional framework has recently been pro- response and protection generated by vaccines available in Chile. To posed as a way to respond to emerging diseases that require a multi- date, 33 injectable vaccines against P. salmonis are commercially disciplinary approach termed the ‘One Health’ approach. Such ap- available, and most are based on inactivated pathogens or recombinant proach recognizes the interdependence of human health, animals and proteins administered via intraperitoneal (IP) injection (4 are subunit ecosystems, but still provides little guidance for researching these in an vaccines, and 29 are inactivated vaccines; 31 vaccines are injectable, integrated manner. An important research gap relates to the role of and 2 are oral) (Rozas and Enriquez, 2014). Despite that IP injection antibiotics used on salmon over human health. In addition, there is a ensures precise antigen dosage to every fish in the culture system with need for research which assesses aquaculture policies in the context of minor vaccine loss, it has some major drawbacks such as the need for an disease risk to humans, wild fish and farmed salmon, and to promote established infrastructure of qualified personnel, the induction of stress the sustainability of the Chilean salmon industry. Policy coherence in in small fish, and in bigger fish, the risk to acquire additional infections aquaculture should be tackled both horizontally and vertically. by the injection point (Tobar et al., 2011). Although they produce “Horizontally”, at country level, government and donors share the re- variable long-term results, all of these vaccines are somehow effective sponsibility for strengthening good governance and inter-sectoral co- in preventing the initial SRS outbreaks that occur after the transfer of herence between different policies, and between stakeholders who may fish from fresh water to seawater for the on-growing stage. Regardless advocate conflicting objectives. The main goal is that policies on fish of their nature and administration route, the vaccine's field results have health, biodiversity, environment, agriculture and others should no not been sufficiently documented and have shown relatively limited longer be in contradiction. It is also critical to measure the impacts and protection, particularly up to 1800 degree-day after injected primo- effectiveness of animal health decisions including cost-benefit analysis, vaccination (Rozas and Enriquez, 2014). Strategies that will enhance cost-effectiveness, welfare measures, externalities, risk, asymmetric the uptake, processing and presentation of the antigen to the fish innate information, strategic behavior, or others. Unfortunately, our literature immune system at the mucosal level, for example, the development and review indicates that there is little knowledge about science-based use of boosters for vaccination strategies, are obvious parallels field of system optimization to reduce fish diseases (only one example for avian research. Finally, the need to define a standard and adequate method(s) and human influenza policies in South-East Asia (Pongcharoensuk to assess vaccine safety, efficacy and transmissibility is an ultimate goal et al., 2012)). There is also very little peer-reviewed information about to document and validate vaccine protection in both experimental and economic costs, information management, and human behavior, in re- field settings for P. salmonis. lation to fish diseases management policies. Policy related articles regarding P. salmonis management are absent 5. So what? Design of a strategy from the literature, and there are no clear evidences that policies are directly related to scientific studies. It is important to develop indicators Situations in which there is little information sharing between sci- for scientific-based norms that can interplay between environmental entists and decision-makers, and scientific information limits decision norms and policies. An example of the lack of scientific-based norms is making, call for a substantial increase in applied and targeted research. the defined minimum interval of 3 months for sampling/testing for P. As salmon farming takes place in changing marine environments, where salmonis per farm or the classification of a farm as a P. salmonis “su- several external factors affect the diseases' incidence and spread, perspreader” whenever half of its cages reports over 0.35% of weekly knowledge from diverse disciplines must be integrated. This knowledge SRS-specific mortality. In both cases, the critical threshold values were must range from cell biology and genetics to environmental disciplines defined by the national fish health authority (Sernapesca) with no clear such as oceanography and ecology. In addition, productive and man- scientific basis or field study to support such limit. It is important to aim agement decision such as pharmacological treatments, productive cycle for policy coherence in the salmon aquaculture sector by accounting for schemes, fish densities and others, must be tested through co-learning stakeholder perceptions to inform the policy-making process. Finally, processes between the industry and scientists. Finally, the “human di- the overall economic costs of SRS and other fish diseases to the industry mension” of fish diseases management, including human behavior, in- and to the country, has not been systematically assessed, which are stitutional frameworks, knowledge management, decision making, important indicators to policy impact assessment and industry deci- economics and policies also determine current status of the diseases in sions. Considering the sanitary costs of SRS at farm and industry levels, the Chilean salmon farming industry. This complex and multifactorial is key to understand the benefits and costs of implementing disease- problem needs the integration of multiple disciplines in a comprehen- related policies. sive interdisciplinary research scheme which could benefit from a Vaccine development against P. salmonis was identified as an im- government led aquaculture research institute which can integrate portant research area (8). The efficient control of and treatment for SRS multiple stakeholder needs and generate cross scale linkages between have been difficult to achieve because there are no efficient commercial actors resulting in significant gains for the environment and the in- vaccines (Leal and Woywood, 2007; Marshall et al., 2007; Tobar et al., dustry and novel cross-sector alliances. 2011), and antibiotics have a limited effect on the disease (Rozas and Enriquez, 2014). Despite positive results for piscirickettsiosis vaccines 6. Conclusions in experimental trials and although more than 10 years have passed since the first vaccine against P. salmonis was launched in the Chilean By the combination of two sources of information (expert opinion market, it is not clear if commercially available vaccines provide full and scientific literature), this study has identified eight research areas protection throughout the production cycle (Maisey et al., 2017). New and a number of research questions that, if developed in the long-term, knowledge about relevant virulence factors is a prerequisite to develop will greatly increase the quantity and quality of new knowledge to an efficacious vaccine against P. salmonis. Because the knowledge about delineate health management strategies to prevent and control the most the most relevant virulence factors is still unknown, developing new important infectious disease that affects farmed salmon in Chile. These vaccines against P. salmonis, still needs to address this research gap. In research areas need to be developed based on an integrative, colla- fact, a limited number of P. salmonis proteins have been characterized borative and crosscutting interaction, aimed to implement a “One for their use as potential candidates for vaccines studies (Marshall et al., Health” approach to successfully control infectious diseases (Osburn

219 F.O. Mardones et al. Aquaculture 482 (2018) 211–220 et al., 2009; Pettersen et al., 2015). In essence, a long term plan will be (3), 215–228. Mardones, F., Perez, A., Sanchez, J., Alkhamis, M., Carpenter, T., 2010. Parameterization able to understand and model the impacts of P. salmonis over the pro- of the duration of infection stages of serotype O foot-and mouth disease virus: an ductivity of the salmon farming industry, the physical and ecological analytica review and meta-analysis with application to simulation models. Vet. Res. environment, the socio-economic sustainability, assess the policy and 41, 45. Marshall, S.H., Conejeros, P., Zahr, M., Olivares, J., Gomez, F., Cataldo, P., Henriquez, V., management implications, as well as provide productive solutions to 2007. Immunological characterization of a bacterial protein isolated from salmonid the industry. Conducting more research on P. salmonis in partnership fish naturally infected with Piscirickettsia salmonis. Vaccine 25, 2095–2102. with the industry, can stimulate the salmon industry to strengthen their Murray, A.G., 2013. Epidemiology of the spread of viral diseases under aquaculture. Curr. – capacity to produce and use such research to guide decision-making, Opin. Virol. 3, 74 78. Oidtmann, B.C., Peeler, E.J., Thrush, M.A., Cameron, A.R., Reese, R.A., Pearce, F.M., improve the efficiency of the production system, increase account- Dunn, P., Lyngstad, T.M., Tavornpanich, S., Brun, E., Stark, K.D.C., 2014. Expert ability in the work of researchers, and contribute substantially to the consultation on risk factors for introduction of infectious pathogens into fish farms. – development of a sustainable industry. This work identifies and tackles Prev. Vet. Med. 115, 238 254. fi Osburn, B., Scott, C., Gibbs, P., 2009. One world - one medicine - one health: emerging key scienti c knowledge and technological transfer gaps for reducing veterinary challenges and opportunities. Rev. Sci. Tech. 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