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“TECHNICAL BASES FOR THE ESTABLISHMENT OF A PROPERLY

CHARACTERIZED NATIONAL PISCIRICKETTSIA SALMONIS STRAIN

COLLECTION”

This is a working synthesis prepared by Acuaim SpA in order

to inform a Consultative Table and was based on the original

Report with same name prepared by the Universidad Catolica de

Valparaiso in February of 2017. The complete Report is available in

Spanish and can be requested directly from the Program Staff.

TABLE OF CONTENTS

INTRODUCTION ...................................................................................................................... 3

GOALS ........................................................................................................................................ 4

METHODOLOGY ...................................................................................................................... 6

CONCLUSIONS ......................................................................................................................... 38

TECHNICAL BASES FOR THE ESTABLISHMENT OF A PROPERLY CHARACTERIZED NATIONAL PISCIRICKETTSIA SALMONIS STRAIN COLLECTION 3

INTRODUCTION

Our contribution within the Program is to provide alternative solutions with high efficiencies

and efficacies for understanding and controlling the facultative intracellular bacteria

Piscirickettsia salmonis, a highly aggressive pathogen that attacks salmonid fish during

their fattening phase in the sea, for which an efficient control and prevention system is

currently lacking. Our goals within the program are: 1) to understand the biology of the target

bacteria, in order to facilitate its early diagnosis and to generate the tools for its control and

eventual eradication; 2) to clarify its epidemiological behavior patterns, in order to use them

as predictive instruments for future epizootic diseases; 3) to understand its relationship with

the environment, clarifying its eventual transmission vectors and/or the role of its passive

or active hosts in the maintenance of its viability, pathogenicity and virulence. Finally, and

as a result, our goal is to establish a national strain collection with the bacterial variants

recovered from farmed fish at various times and latitudes, facilitating its biological and

epidemiological monitoring and reducing its impact on future productivity.

TECHNICAL BASES FOR THE ESTABLISHMENT OF A PROPERLY CHARACTERIZED NATIONAL PISCIRICKETTSIA SALMONIS STRAIN COLLECTION4

GOALS

General goals

1. To define an operative platform that allows for the establishment of an official, reliable

and sustainable P. salmonis strain collection that includes the main variants/strains

recovered from the three salmonid species farmed in Chile as a basis for the application of

more effective sanitary management.

2. To conduct fundamental research that helps clarify the crucial parameters of P. salmonis’

biology associated with the development of the disease and its control.

Specific goals

An official Piscirickettsia salmonis strain collection.

1. To define the optimal growing conditions for P. salmonis.

2. To define a more efficient procedure for the isolation of P. salmonis.

3. To characterize the P. salmonis isolates/variants/strains by means of massive sequencing

and phylogenetic and metagenomic analyses.

4. To isolate P. salmonis variants by geographic area and species, for the years 2016-2017.

5. To establish management strategies for the sustainability of the Official Strain Collection,

in its first phase.


Piscirickettsia salmonis’ biology

1. The evaluation of the role of water salinity in the viability and virulence for P. salmonis

(in vitro/in vivo).

2. To characterize P. salmonis isolates by degree of pathogenicity and virulence (in vitro/in

vivo).

3. Screening of potential biological and mechanical vectors or intermediary bacterial hosts.

4. The identification of the mechanisms involved in vertical transmission.


METHODOLOGY

In order to achieve these goals, various research goals with associated activities were defined.

Defining the optimal growing conditions for P. salmonis.

Activity 1. Reactivation of cryopreserved variants/strains

Cultivation, storage and reactivation protocol specific for P. salmonis was prepared, and is

explained in figure 1. In order to develop this protocol, all of the cryopreserved Piscirickettsia

salmonis variants/strains preserved in the Institute of Biology of the Pontificia Universidad

Católica de Valparaiso, more specifically in the Laboratory of Genetics and Molecular

Immunology (GIM-PUCV), were sequenced along with variants that had been preserved

since 2008 in the Institute of Biochemistry and Microbiology of the Universidad Austral de

Chile (UACh). Each of these variants/strains underwent the procedure detailed in figure 1,

containing 3 steps.

Of all of the stored P. salmonis variants (50), 28 underwent reactivation, approximately 34%

of which were recovered and registered as viable (V), 22% were not recovered and thus were

registered as non-viable (NV), and the rest of the stored variants are still undergoing the

reactivation procedure, catalogued as pending (figure 4). The variants that were recovered,

among them the reference strain ATCC LF89 VR 1361, are detailed in table 1.

Figure 4. P. salmonis variants percentage subjected to reactivation.


Table 1. Variants of P. salmonis cryopreserved until 2015, subjected to reactivation

P.salmonisVariants P.salmonisVariants

Viable Non-viable Pending Viable Non-viable PendingLF89 AUS 004 AUS 019 AUS 044 AUS 047

AUS 002 AUS 006 AUS 020 AUS 045 AUS 049AUS 005 AUS 009 AUS 021 AUS 046 AUS 053AUS 007 AUS 010 AUS 023 AUS 048 AUS 054AUS 008 AUS 012 AUS 024 AUS 051 AUS 055AUS 025 AUS 013 AUS 029AUS 036 AUS 015 AUS 030 AUS 052 AUS 056AUS 037 AUS 016 AUS 031 AUS 057AUS 040 AUS 017 AUS 032 AUS 058AUS 041 AUS 018 AUS 034 AUS 059AUS 042 AUS 050 AUS 038 AUS 060AUS 043 AUS 038

The growth in Austral SRS liquid environment of all of the recovered (viable) variants was

evaluated and sent in solid environments to the strain collection in the Pontificia Universidad

Católica de Valparaiso. However, another percentage (44%) of variants is still undergoing

the reactivation procedure.

When the variants were received at the GIM-PUCV, the different strains were seeded in liquid

environments until growth was observed, and they were afterwards reseeded on Agar plates.

In parallel to this procedure, cell cultures (SHK-1) were directly infected with vials containing

variants/strains from -80°C. Once ECP was shown, the supernatant was transferred to a

liquid environment and then to agar plates.

The results of the recovery of variants/strains obtained from the GIM-PUCV are shown in

table 2.


Table 2. Variants/strains recovered at the GIM-PUCV detailing origin, species and tissue,

indicating current viability.

Origin Isolate Organism Organ Currently Viable

1 PUCV LF-89ONCORHYNCHUS

KISUTCHX

2 PUCV EM-90 SALMO SOLAR X

3 PUCV TGIM-11ONCORHYNCHUS

KISUTCHHIGADO X

4 PUCV TGIM-12 SALMO SOLAR CAVERNA X

5 UACH AUS 040 SALMO SOLAR


7 UACH AUS 061 SALMO SOLAR RIÑÓN

8 UACH AUS 008ONCORHYNCHUS

KISUTCHRIÑÓN









17 UACH AUS 071 SALMO SOLAR RIÑON/HIGADO

18 UACH AUS 072 SALMO SOLAR RIÑON/HIGADO

19 UACH AUS 088

20 UACH AUS 089




23 UACH AUS 093ONCORHYNCHUS

KISUTCHRIÑÓN

24 UACH AUS 002

25 UACH AUS 005

26 INTA CRG1

27 INTA CRG2

28 INTA INTA1

Activity 2. Evaluation of various culture media free of cells, solids and liquids

PUCV: Kinetics of growth in different media. In order to carry out this comparative evaluation,

the use of recommended protocols from the manufacturers of each medium was established.

Liquids:

• BM3 (Henriquez et al., 2013. A Novel Liquid Medium for the Efficient Growth of the

Salmonid Pathogen Piscirickettsia salmonis and Optimization of Culture Conditions.

PLoS ONE 8(9): e71830)

• AUSTRAL (Yañez et al., 2012. Broth medium for the successful culture of the fish pathogen

Piscirickettsia salmonis. Dis Aquat Org 97:197-205

• P. salmonis culture liquid according to the IFOP formula shared with the laboratories

participating in the MIC to antibiotics Ring Test.

• Müeller-Hinton liquid culture adjusted cations (Diagnotec).

Solids:

• Austral (Yañez et al., 2013. Two novel blood-free solid media for the culture of the salmonid

pathogen Piscirickettsia salmonis. Journal of Fish Diseases 36: 587–591) y

• BCG or blood (Mauel et al., 2008. Culture of Piscirickettsia salmonis on enriched blood


agar. J. Vet. Diag. Invest. 20: 213-214

• Heart cysteine + hemoglobin Agar plates (Diagnotec).

• Enriched blood Agar plates (Diagnotec)

• IFOP’s solid agar medium

Figure 7. Growing curves of different isolates of P.Salmonis grown at 18 °C in liquid culture media presenting the better results: (A) AUSTRAL and (B) BM3.

UACh: Kinetics of growth in different media. Growth curves were calculated for certain

variants in which 4 different culture media were used, including 2 previously documented

media.

• Austral SRS broth (SRSb) (Yañez et al., 2012)

• BM3 (Henriquez et al., 2013)

The two remaining media are enriched basal media:

• Marine Broth (MB)

• Medium X (MX).


Figure 8. Growth curves of different variants of P.samonis in different media

Based on experience obtained from prior tests, it is impossible to define just one culture

medium for P. salmonis growth, without taking into account that there is a large variety of

isolates that respond differently to the available media. Therefore, it is necessary to have a set

of media formulas free of cell in order to evaluate each isolate, choosing the most adequate one

for each. However, an important characteristic to take into account when deciding regarding

which medium to use is the composition of each medium, given the cost implied in creating

each one. In this sense, the IFOP medium seems to be the most costly because of its contents:

fetal bovine serum and defibrinated blood.


Activity 3. Evaluation of growth and infectivity in different cell lines.

In order to carry out this activity, we defined various cell lines based on the scientific

information available. The first cell line is SHK-1, derived from cephalic kidney in Atlantic

salmon, the preferred cellular type for bacterial development, as well as RTgill-W1, a cell line

derived from the gills of Oncorhynchus mykiss trout, of epithelial type used for toxicity and

virology studies in salmon.

P. salmonis infectivity in two salmon cell lines using two strains was compared: LF-89, the

strain type and C24, a natural isolate. Figure 9 shows the expression in cell line SHK1 for

virulence marker genes ChaPs and dotH, and figure 10 shows RTgill’s expression.

Figure 9. Early time expression of infection in SHK1 Cellular line. A: ChaPs and B: dotH.

Figure 10. Early time expression of infection in RTgill Cellular line. A: ChaPs and B: dotH


Definition of a more efficient method for P. salmonis isolation.

Activity 1. Evaluation of cryopreservation methodologies (preservation and reactivation).

Three cryopreservation methods were used based on three cryopreservants (table 5).

Table 5. Cryopreservants choice. Methodologies used for cryopreservation tests in variants/

strains of P.salmonis.

Method 1 Sucrose at 15% + Glycerol 25%

Method 2 SBF at 20% + Glycerol at 20%

Method 3 SBF at 20% + DMSO at 10%

Each one of these methods was tested in different P. salmonis variants in order to compare

other variants that had undergone the same cryopreservation process; however these variants

are still in the testing process,

In the freezing procedure, methodology 1 extracts the quantity of the culture that is to be

cryopreserved and adds the saccharose-glycerol stock to it in a 1:1 dilution, so that the final

concentration is 15% saccharose and 25% glycerol.

For methodology 2, the quantity of the culture that is to be cryopreserved is extracted and

glycerol and SBF is added so that the final concentration of both, when separated, is 20%.

For methodology 3, the quantity of the culture that is to be cryopreserved is again extracted,

and first the SBF is added so that the final concentration is 20%, and finally the DMSO is

added so that the final concentration is 10%.

The viability of the P. salmonis variants was evaluated with the three different methodologies.

With methodology 3, distinct concentrations of DMSO were also evaluated, based on the

centrifugation of the liquid culture and on the number of bacterial cells to be cryopreserved.


Activity 2. Optimization of growth media for natural isolates.

In order to carry out this activity, the same isolates used to determine the kinetics of infection

in activity 2 were used, and certain characteristics of the media were modified in order to

obtain more efficient results.

Among the parameters that were modified are the following:

• Temperature

• Gelling agents

• Reducing agents

Temperature: In accordance with the aforementioned conditions, the optimal temperature

for the growth of P. salmonis was evaluated, varying the temperature between 18°C and

20°C, given that these are the temperatures that are most widely used in laboratories where

cultivation is carried out. The growth curves for the isolates in different culture media

(Austral, BM3 and IFOP) were obtained. However, issues regarding the reproducibility of

growth of the isolates appeared, demonstrating the need to further study this growth with a

greater quantity of replicas of the tests, using all of the isolates of the strain collection. For

now, this has not been achieved due to the fact that the obtained results are disparate and

unreliable; or rather there are isolates that have not been grown in liquid media, but only on

plates. These factors are indicators of the complicated issue of maintaining P. salmonis in a

laboratory.

Gelling agents: For solid culture media, Agar is generally used as a gelling agent. However,

we tried switching it with Gelrite, because it allows for a better availability of nutrients on

the plates for the bacteria. Tests with Piscirickettsia salmonis LF-89 seeding were carried

out on different plates, with agar and Gelrite, and with BCG plates (blood), with different

concentrations of Gelrite, injected with the same strain.

The best solidity was achieved with 3gr/L of Gelrite, this being the most optimal surface for

the homogeneous seeding and growth of the bacteria.


Reducing agents: In order to test the impact of the reducing or chelating agents on the

growth, different concentrations of DTT were added to the liquid and solid cultures in order

to evaluate the growth of P. salmonis.

Figure 21. Piscirickettsia salmonis growth curves under different concentrations of DTT (A) 0,5 g/L and (B) 1 g/L, added to BM3 media.

Activity 3. Developing an operational and standardized manual that contains procedures for the sampling, isolation, growth and maintenance of P. salmonis in laboratories.

Laboratory manuals are guides for practical procedures that can be carried out in laboratories,

serving as technical and theoretical support. They must contain the specific and standardized

procedures that allow for adequate use of equipment and for the development of new protocols,

which can also be used in training and updating processes. In this context, the operative

manual will be added to during the project, with the goal of validating and officializing it by

the end of the project, at the end of 2018.

The aim of the manual is:

• To establish standardized procedures that must be applied for the extraction, manipulation

and obtaining of fish, organs and/or fish fluids that will be used as biological samples for

the isolation of Piscirickettsia salmonis (PS).

• To establish standardized technical protocols for the isolation, growth and maintenance

of Piscirickettsia salmonis (PS) isolates.

• To establish the biosafety measures for the location where sampling and isolation are

carried out, as well as for the involved personnel.


The manual takes into consideration the following issues:

1. Personnel

2. Materials

3. General Biosafety

4. Sampling procedures

5. P. salmonis isolation

6. Maintenance of P. salmonis in the laboratory

Characterizing P. salmonis isolates/variants/strains through massive sequencing, bioinformatics and phylogenetic analyses.

Activity 1. Validated sequencing of 16S and ITS markers.

The following steps were used for the sequencing of these markers:

1. Obtaining the genomic DNA

2. PCR for amplifying 16s and ITS genes from the ribosomal operon

3. PCR product cloning for ITS and 16s

4. Sequencing and analysis of PCR products for the ribosomal operon

5. Bioinformatic analysis

The sequence obtained for the 16S ribosomal gene is approximately 1535 pb. Therefore direct

sequencing was applied for this particular case, and for the purposes of the sequence analysis,

ClustalW and phylogeny analysis, the sizes of the sequences were adjusted in order to avoid

a distortion in the results.


Activity 2. Structuring the phylogenetic analysis of sequenced variants.

Among the described methodologies for the molecular and genomic characterization of

bacteria is the PCR application, designed to amplify specific DNA fragments. The 16S ribosomal

gene and the intergenic spacer are the most recurrent targets for this characterization.

Ever since the 70’s and the end of the 80’s, these have been used for the identification and

phylogenetic characterization of the different types of prokaryotes. This methodology is

quick, very exact and is nowadays facilitated by the access to databases and software that

allow for rapid phylogenetic analyses. In this sense, there are thousands of sequences of 16s

rDNA and of the ITS intergenic spacer for all of the known genders of bacteria.

We addressed the joint genomic characterization of all of the isolates of the strain collection,

with isolates originating from different massively-farmed salmonid types, different

geographical regions in the south of Chile and with different salinity conditions and water

types.

In order to carry out the genomic characterization of all of the isolates, the following steps

were followed:

1. Bacterial DNA extraction

2. PCR for the identification of P. salmonis

3. PCR for the 16S and ITS ribosomal genes in P. salmonis

4. Purification of PCR products

5. Ligation to the pGEM-T Easy® cloning vector

6. Transformation with the ligation product and clone selection

7. DNA sequencing

A phylogenic analysis of all of the isolates of the strain collection was carried out, including

strains from the UACh that are not included in the mirror strain collection. Some of these

strains catalogued with the initials IBM have been available for two years, such as 16S


sequences and ITS sequences in the DNA database of the NCBI. It is interesting to highlight

the fact that these IBM strains were previously used for the PCR-RFLP analysis of the 16S

fragment by Mandakovic et al. (2016), and through this analysis it was concluded that the

national strains are distributed into these two geno-groups or geno-types: one group of

bacteria is similar to the LF-89 strain and another group of bacteria is similar to the EM-90

strain. The result obtained from the phylogenetic analysis of the DNA sequences of the 16S

ribosomal gene clearly show the formation of two main genogroups, LF-89 type and EM-

90 type, as is shown in figure 26. In addition, the external position of both genogroups in

the 16 S sequence for the Franciscela piscicola species is highlighted, this external location

validating the structure of the generated phylogram.

Figure 26. Phylogenetic tree obtained by Bayesian inference showing the evolutive position of the different strains of P.salmonis. The number next to the nodes indicates the later probability for the Bayesian analysis. The scale represents the genetic distance in nucleotides substitutions by site. The analysis was done with the PAUP and Mrbayes tools. Strains EM-90 and LF89 were remarked (AUS 089)

Based on the analysis of the sequences of 16S and ITS, the analysis of various P. salmonis

isolates and strains coming from different geographical zones from the south of Chile clearly

shows, with regards to this last marker, that these strains are distributed into two large

genogroups, one of which contains the LF-89 reference strain, while the other contains

another national strain, EM-90. This distribution seems to indicate that the majority of the

strains that are contained and analyzed in this study match or can be derived from these two

“ancestral” or firstly isolated strains in the country. The phylogenetic analysis of ITS clearly


shows that there are strains that do not show any homology with either genogroup, forming

independent genogroups.

No correlation between genogroups regarding the geographical origin of the strains was

found, nor was any correlation with the water type (for example, seawater or estuary water),

as was the case with the host fish from which the pathogen’s strain was isolated. This result

is compatible with the results obtained by Casanova et al. (2003) and with the analysis carried

out by Otterlei et al. (2016) that compared Chilean strains to Canadian ones.

Activity 3. Sequencing and validation of 20 variants/strains.

With the objective of carrying out an integral characterization of the different P. salmonis

isolates that compose the strain collection, 20 genomes were sequenced in order to allow for

an analysis determining the genetic levels of the main differences and similarities between

the different isolates, including shared genes (core genome) and individual genes or those

belonging to each isolate or strain.

For all of the sequenced isolates, it was possible to determine the presence of 4 circular

plasmids that differ in size (table 8). Generally speaking, these extra chromosomal elements

are relatively conserved in size among the different isolates. It is important to emphasize

that, given the fact that all of the genomes have a large quantity of mobile elements,

repeated sequences and ISs insertion sequences, it was relatively complicated to assemble

and circularize the chromosomes and plasmids. Finally, by means of PacBio and Ilumina

sequencing tools, it was possible to obtain high quality sequences, with a final score of QV60,

corresponding to 1 error per 1 million base pairs.


Table 8. Summary of the results obtained by the sequenciation of 20 isolates of P.salmonis.

Isolate Chromosome Number

Chromosome Size

Plasmids Number Plasmid Size Genome Status

GIM-001 1 3,1 Mb 4 pendiente borrador

GIM-005 1 3,1 Mb 4 110 kb, 80 kb, 37 kb, 24 kb completo





GIM-014 1 3,1 Mb 4 194 kb, 57 kb, 38 kb y 33 kb completo




AUS-007 1 3,1 Mb 4 pendiente borrador







This is the first analysis that has been carried out for the complete genome of various P.

salmonis isolates. The successful completion of the chromosomal sequence of the 20 isolates

sent out for sequencing was possible, but due to the existence of multiple transposon type

sequences, it was not possible to circularize all of the plasmids. The size of the chromosomes

is within the range of 3,1 Kb (3,1 million base pairs).


The phylogenetic analysis of the completed genomes and the inclusion of 19 additional

genomes from the Genbank database allowed for the corroboration of the previous results, in

which two apparent genogroups were found, one of these being related to the LF-89 strain,

and the other with the EM-90 strain. In this context, all of the completed isolates belong to

the EM-90 genogroup.

Isolating new P. salmonis variants per geographic area (Aysén and Los Lagos Regions) and per species, years 2016-2017.

Activity 1. Establishing a procedure for sampling that includes a specific protocol and requirements that farming centers must meet in order to carry out sampling within the framework of this project.

The work methodology was based on interviews with the persons in charge of specific

procedures (sampling/culture) in each entity, where we collected the different experiences

accumulated over many years of work with this bacterium.

The results of this activity were included, for practical reasons, in the “Operative and

standardized manual for the sampling, isolation, growth and maintenance of P. salmonis in

laboratories”, previously presented.

Activity 2. Isolation of new P. salmonis variants/strains from field samples, years 2016-2017, in at least 40 farming centers located in the Los Lagos and Aysén Regions.

The majority of these isolations were completed by IFOP, the entity that established the

cooperation agreement. Based on this agreement, and with the help of SERNAPESCA, 36

samplings were carried out in farming centers that presented Piscirickettsiosis problems,

between December 2016 and June 2017.

At the end of this report the IFOP had realized a total of 36 samples, of which 26 isolates

have been recuperated and sent to the GIM-PUCV. The report delivered by the IFOP and more

specifically the information regarding sampling has been added in the annexes of this report.


A total of 42 natural isolates have been received to date, summarized in table 9 together with

the information sent in the technical information sheets, where IFOP’s contributions to the

Official Strain Collection is detailed, among other contributions, as well as the actual status

of said isolates/strains.

Table 9. Natural isolates received from IFOP, including species, organ and geographic location

of the isolate.


Establishing management strategies for the Official Strain Collection’s sustainability.

Activity 1. Designing a proposal for strategic guidelines

Based on a complete review of the information currently available concerning the

sustainability mechanisms of the existing strain collections in the world, a framework

proposal was created, which was reviewed and adapted to the Chilean situation and to the P.

salmonis situation. Likewise, we defined the modality or strategy to be implemented in order

to support the Official P. salmonis Strain Collection over time, together with the participating

entities and an external consultant, Professor Overmman from the DSMZ, one of the most

important strain collections in the world.

Definition: The strain or microorganism collections are sources of genetic resources,

whose purpose is the preservation of biological diversity, guaranteeing its availability for

educational, research and commercial activities, when applicable.


Purpose: The main purpose of a microorganism collection, also known as a strain collection,

is to preserve its viability, along with its biochemical, immunological and genetic features.

In this case, it is also crucial to understand the geographical distribution of the bacterial

variants, in order to contribute to a better understanding of its epidemiological patterns in

national farming centers.

It is also important to emphasize the fact that the organization in charge of the Strain

Collection will represent an official link between the national and international scientific

community and the laboratories associated with the Sernapesca network, in order to favor

access to validated bacterial strains and variants, and to contribute to the field of bacterial

biology and epidemiology in the national aquatic environment, benefiting the sustainability

of the aquaculture sector.

All of these activities have been carried out with the full understanding that the generation,

maintenance and collectivization of the strains and variants that will constitute the Strain

Collection, are non-profit.

Generally speaking, the persons responsible for the Strain Collection will constantly be

concerned for all of the aspects of the bacterial variant collection activities, and in particular

they will encourage the development of new initiatives that favor the understanding and

control of the involved microorganism and the improvement of the standards of scientific

services provided for the national and international community of users.

Activity 2. Workshop and discussion/coordination meeting for the establishment of a regulatory framework that will guide the functioning of the Official Strain Collection

On June 16th 2017, in the facilities of the Curauma campus of the Pontificia Universidad

Católica de Valparaiso (PUCV), specifically in the Institute of Biology, a thematic workshop

was carried out in the context of the FIE 014 Project, with the participation of the director of

the FIE projects, part of the UACh team and the whole team from GIM-PUCV. This workshop

had two main parts, one of them concerning the project’s advances and the other concerning

the continuation of the Strain Collection once the project finishes.


Participants:

SERNAPESCA –FIE PROJECT GIM-PUCV GIM-PUCV

Fabián Avilés Soraya Díaz

Dr. Sergio Marshall Dr. Fernando Gómez Dr. Jimena Cortez Dr. Nicolas Ojeda Gabriela Carril Dannia Gimenez

Dr. Alejandro YañezDr. Denise Haussmann

Activity 3. Definition of communication strategies for the diffusion and sustainability of the Official Strain Collection.

• It was defined that the communication strategy would be mainly focused on:

• A web page that would portray the Strain Collection and its features, hosted in the FIE

project’s main web page.

• Indexed scientific publications with the largest amount of results possible obtained within

the framework of the project.

• Presentations in seminars, congresses and in courses regarding this field.

In vivo/in vitro evaluation of the role of water salinity in the viability and virulence of P. salmonis.

In vitro evaluation: Using the samples of organs extracted from the in vivo evaluation,

cell cultures were infected in order to evaluate the bacteria’s virulence, by measuring its

replicative rate, determining the cytopathic effect and differential expression of virulence

markers by means of RT-qPCR.

The growth of three strains chosen arbitrarily from the National P. salmonis Strain Collection

were evaluated in two different salinity conditions (0,8% and 1,5%) and in the same Austral-

SRS medium conditions. This showed that there are practically no differences in bacterial

growth in the analyzed conditions. However, this does not dismiss the fact that the bacteria


do show some differences in genic or virulence expression, which must be analyzed in the

future.

Following this procedure, we quantified the degree of pathogenicity of the P. salmonis strains

in the SHK-1 cellular line, visualizing the cytopathic effect in the two salinity conditions. It

was demonstrated that both field strains present a markedly higher cytopathic effect than

the control LF-89, but it is difficult to estimate the difference in growth for both salinity

conditions. As a control, an uninfected culture in which there is no cytopathic effect was

observed.

In vivo evaluation: The goal of this evaluation was to carry out a test with two P. salmonis

isolates in Atlantic salmon (S. salar) experimentally infected by cohabitation in two different

salinities.

The mortality/survival rates caused by the 02/13 and AUS/005 P. salmonis isolates were

determined for Atlantic salmon (S. salar) in cohabitation, as well as the mortality/survival

rates of the 02/13 and AUS/005 P. salmonis isolates in tested Atlantic salmon for cohabitation

in two different salinities.

Figure 38. Accumulated mortality curves of the challenge with two strains and two salinity conditions. The figure shows the different behaviors of both strains in relation to the accumulated mortalities during the entire essay. Is easy to observe the differences in the mortalities generated by both strains. While AUS 0213 strain showed similar mortalities to both salinity conditions, the AUS 005 strain showed clear differences by a sharp and fast increase in 5% salinity.

Accumulated mortality of Salmo salar challenged by two isolates of P.salmonis

and 2 salinities.

Mor

tali

ties

per

cen

tage

Grupo control

AUS 005 20% Salinities




Days


This result indicates that Atlantic salmon in 5% salinity tested with the AUS005 P. salmonis

isolate showed a higher mortality rate than fish in a 20% salinity rate with the same isolate.

The test by cohabitation with P. salmonis 0213 at 20% behaved according to the standard,

with mortalities in a range between 50 and 60%.

Characterizing P. salmonis isolates by degree of pathogenicity and virulence (in vitro/in vivo).

The P. salmonis strains analyzed in this activity were the strains that had massively sequenced

and partially assembled genomes (approx. 85%). For the purpose of this analysis, only the

larger contigs were taken into account in order to consider only the eventual pathogenicity

islands or integral genomics.

In order to evaluate their pathogenicity, a PIPS: Pathogenicity Island Prediction software

was used in Default mode and without any modifications to the parameters for all of the

predictions. The various strains that were studied were used in an Embl format (table 12).

Table 12. Genomic or pathogenic islands found in field strains of P.salmonis. Field strains

have more islands than the reference LF-89.

Strain Host Nº de IG

LF-89 O. kitsutch 0*

AUS040 S. salar 12

AUS041 O. mykiss 12

AUS042 S. salar 10

AUS007 O. kitsutch 6

IBM023 S. salar 14

AUS043 S. salar 9

AUS044 S. salar 9

AUS045 O. mykiss 10

AUS025 O. kitsutch 15


Based on this bioinformatic analysis, it is possible to complete the research and analysis of

the genomic islands in each one of the genomes of these strains. In addition, it is interesting

to note that another goal for this project is the massive sequencing of 20 genomes for P.

salmonis field strains. Thus, based on the results of this analysis, it will be possible to obtain

relevant information from the genomic islands present in P. salmonis.

In order to evaluate the resistance to antibiotics, ELISA plates were used in concentrations

from 0,02 μg/mL to 8 μg/mL. Absorbencies of 600nm were detected in the microplate reader,

using the associated software for analyzing data, which was afterwards graphed using

SigmaPlot 11.0 software (table 13).

Table 13. Minimum Inhibitory Concentrations for national strains of P.salmonis. Liquid

medium culture at 18 °C for 5 days. The remarked strains show “old” strains isolated in the

’90, that is, close to LF-89 and/or EM-90 strains.

Strain Florfenicol (μg / μL) Oxitetraciclina (μg / μL)

AUS040 0,250 0,8

AUS041 8 1

AUS042 1 0,125

AUS023 4 4

AUS043 4 0,5

AUS044 1 0,25

AUS025 2 1

AUS045 0,250 0,125

LF-89 0,250 0,125


When observing this analysis, it is clear that all of the field strains studied require a higher

concentration of antibiotics in order to be inhibited regarding the LF-89 strain. On the

other hand, the studied strains presented a higher susceptibility to oxytetracycline than to

florfenicol, probably due to the massive use of the latter in the national salmon industry.

Lastly, it was found that the AUS045 and AUS040 are the oldest strains from the UACh strain

collection, both having been isolated in the 90’s, and are therefore highly susceptible to

antibiotics in comparison with the more recent isolated strains.

For the in vivo evaluation, a trial was carried out in which the expression for two virulence

markers in the bacteria was evaluated through qRT-PCR. The first was the gene that

codifies the immunogenic ChaPs (HSP60) protein, which is involved in the invasion process

(Marshall et al., 2007). The second marker was the gene of the DotH protein, which is part of

the bacteria’s Dot/Icm Secretion System, whose function is to participate in the formation of

the bacteria’s replicative vacuole and intracellular multiplication (Gómez et al,. 2013; Labra

et al., 2016).

The results show that the expression of the dotH marker increases over time in 40% of the

analyzed samples, reaching its highest levels in one of the initial mortalities, indicating that

this marker is actively involved in the infective process (figure 40).

Figure 40. dotH expression in fish challenged with P. Salmonis LF-89 in fresh water.


For ChaP.s, a similar tendency to the one presented by dotH was observed, although the

expression levels are slightly lower (figure 41).

These preliminary results indicate that the ChaPs and DotH markers are useful tools for

validating the infection and the degree of virulence of the bacteria. In order to verify this

observation, an analysis of these same markers in samples obtained from naturally infected

fish (Piscirickettsiosis outbreak) in the Aysén Region during the first trimester of 2017 was

realized. The RNA extraction methodology, cDNA and qPCR syntheses were described above.

It was found that 37% of the samples that were positive for P. salmonis showed an increase in

the expression of the dotH gene (figure 42).

Figure 41. ChaP.s expression in fish challenged with P. Salmonis LF-89 in fresh water.

Figure 42. dotH expression in fish naturally infected with P. Salmonis.


Figure 43. ChaP.s expression in fish naturally infected with P. Salmonis.

Screening of potential biological and mechanical vectors or intermediary hosts for Piscirickettsia salmonis.

Activity 1. Sampling, filtration and DNA extraction.

Sampling was carried out in the Aysén Region, in a geographic zone with farming centers

affected by P. salmonis outbreaks in the month of March 2017. In order to prepare a complete

characterization of the different unicellular eukaryotes, the sampling was realized in the

different biomes associated with the farming centers.

There are samples taken from a cage affected by Piscirickettsiosis, as well as samples taken

from a distance of 100 meters from the farming center. In both cases, the sampling points

were the following:

a. Neuston sampling: 40 liters of surface water were taken from two different points: a)

from the interior of the cages; and b) from a distance of 100 meters from the farming

center.

b. Water column sampling: 40 liters of water were taken from 40cm, 4m and 10m of

depth using a Niksin sampling bottle , at two sampling points: a) inside the cages; and

b) at 100 meters from the farming center.


c. Marine sediment sampling: Using a dredge, a sample of 1 kg of marine sediment from

under the farming center was taken, which will be recovered in a 1L Schott bottle.

d. Fouling sampling: A 1 meter piece of rope from the farming center was taken, from

which all of the attached fouling was removed and used as raw material for the DNA

extractions.

Following the sampling, the water samples from all of the points were subject to filtration,

passing through a circuit of filters of 1 μm and 0,45 μm. The material retained by the 0,45 μm

filters was recovered and put through total DNA purification, using the PowerWater ® DNA

Isolation Kit (Mo Bio) system, in accordance with the manufacturer’s indications. For the

sediment samples, DNA purification was carried out with the PoweSoil® DNA Isolation Kit

(Mo Bio) system, in accordance with the manufacturer’s indications. The purified DNA was

quantified using spectrophotometry in Nanodrop-1000 equipment and kept at -20 until its

use.

Activity 2. Marker amplification.

Once the DNA was obtained from the water and sediment samples, the samples were analyzed

using PCR in order to detect the presence of bacterial and eukaryotic organisms.

For the detection of bacteria, the PCR technique was used with universal primers for 16S,

denominated EubA and EubB, which allowed for the amplification of the rRNA 16S sequence

for the Eubacteria group (Suzuki & Giovannoni, 1996).

In addition, a PCR technique was used for P. salmonis detection in all of the samples, for

which RTS1 and RTS4 primers were used, aimed to amplify the intergenic region or the ITS of

the ribosomal operon (Marshall et al., 1998).

For the detection of eukaryotic organisms by means of PCR, a set of primers directed at the

rRNA18S sequence was used, denominated EukA and Euk1 (Wang et al., 2014).

In the universal 16S PCR of the water and sediment samples, positive amplifications were

obtained in all of the samples coming from water, with the exception of the sample from

40 meters of depth in the location outside of the farming center (at a distance of 100 meters


from the net-cage). On the other hand, both sediment samples resulted negative for the PCR

technique, which is indicative of the fact that the bacterial load in these anaerobic conditions

is very low.

In the P. salmonis ITS PCR of the water and sediment samples, the results for all of the samples

were negative, which indicates that the P. salmonis charge is low or null in the water column,

even when an outbreak of the disease was presented (figure 46). The sediment samples were

not analyzed by means of PCR for ITS, because they had previously been negative for the

bacterial 16S. A probable explanation for the lack of detection of the pathogen in the water is

that antibiotics had been applied in the weeks prior to the sampling.

In the eukaryotic 18S PCR of the water and sediment samples, a positive result for the

amplification of all of the water samples was obtained, in which a product of approximately

1600 pb was generated. For the two sediment samples, no positive results for amplification

were observed, which is probably due to the fact that it is a medium with a very low oxygen

level.

Activity 3. Sequencing and metagenomic analyses and pilot evaluation of co-cultures.

Due to the fact that other bacteria that are phylogenetically close to P. salmonis, such

as Legionella pneumophila and Brucela suis, are able to exist and replicate themselves in

protozoa and other amoeba in water sources, using them as reservoirs, this activity’s goal

is to determine whether P. salmonis uses this same mechanism for survival in marine

environments.

In order to achieve this goal, it is important to identify which are the predominant unicellular

eukaryotes in the salmon farming centers. As an additional and complementary activity,

predominant bacterial genders will also be determined for this medium.

Once the results from the amplification of markers are obtained, the material will be sent to

be sequenced and to prepare a metagenomics of the results, in order to define the possible

microorganisms that are present and most abundant in the samples. Finally, this information

will be used to realize co-culture tests in microalgae in order to evaluate the bacterial presence

in these microorganisms.


The results of this activity are for the most part completed and are presented below. However,

the large quantity and diversity of the organisms found in the samples have caused some

delays in the data analysis. Notwithstanding the foregoing, documents sustaining the above

will be presented.

Identification of the possible mechanisms involved in vertical transmission and verification strategy design.

Activity 1. Identification of vertical transmission mechanisms.

A systematic search that identified the existing and viable vertical transmission mechanisms

in a fish farming system was realized, and a document explaining the possible mechanisms

was prepared.

Despite the fact that P. salmonis’ horizontal transmission has been verified and widely

described, there still have not been any validating studies demonstrating the vertical

transmission of the agent. In 2003, experimental vertical transmission in ova and fry coming

from infected parents was reported (Larenas et al., 2003).In this study, the presence of P.

salmonis was detected in fry when one or both progenitors were inoculated, even though

neither of the infected fry presented signs of the disease. In addition, the bacteria was also

detected in the offspring obtained through fertilizing eggs from non-inoculated females

incubated in a medium containing bacterial suspension, suggesting that infection occurs

during the fertilization process. All of the above analysis was carried out in controlled

laboratory conditions, which is why it is still necessary to validate this mechanism in the field.

In addition, the presence of the bacteria in ovarian fluids and seminal liquid from infected

progenitors must be corroborated, as well as in reproductive organs.

For pathogenic organisms phylogenetically close to P. salmonis (Legionella pneumophila,

Coxiella burnetii and Francisella tularensis), which also have similar infection and virulence

mechanisms, vertical transmission has not been described. This mechanism has only been

described for Brucelosis, a disease that affects ovine, porcine and caprine livestock, whose

etiological agent is Brucella abortus, B. suis and B. melitensis. The vertical transmission of


Brucelosis has been validated since 1971, in a study that demonstrated that 60 to 70% of

fetuses born from infected mothers are also born with the infection (Plommet et al., 1971).

Activity 2. Evaluation of the identified mechanisms associated with P. salmonis.

Taking into account the precedents presented in the previous activity, as well as the complex

growth and maintenance features for Piscirickettsia salmonis, we believe that it is improbable

that this disease is transmitted vertically between the progenitors and their offspring.

However, this hypothesis must be proven empirically in order for it to become a foundation

for policy decision-making.

In order for this to happen, we must discard or confirm the following:

• The egg is infected during its production process in the ovary. For this, it is important to

demonstrate that the bacteria is present and in an infective state in the ovarian tissue.

• The egg is infected on contact with the ovarian fluid. For this, it is important to demonstrate

that the bacteria is present and in an infective state in the ovarian fluid.

• The intra-ova presence of the bacteria following the spawning process. It is important to

demonstrate the presence of the bacteria in an infective state in the egg.

• The extra-ova presence of the bacteria following the spawning process. It is important to

demonstrate the presence of the bacteria in an infective state attached to an egg.

• Presence of the bacteria in a hatched egg (yolk sac fry). It is important to demonstrate the

presence of the bacteria in an infective state in a yolk sac fry.

• Presence if the bacteria in fry. It is important to demonstrate the presence of bacteria in

an infective state in fry.


Activity 3. Definition of verification strategies for the selected mechanisms.

A strategy for the empirical verification of the vertical transmission mechanisms identified

in the above point was defined and proposed.

Hypothesis:

1. Salmonid females infected with Piscirickettsiosis can generate ova that are carriers of

viable and infective P. salmonis forms.

2. Salmonid females infected with Piscirickettsiosis are capable of generating fry that are

carriers of the disease.

Specific goals:

1. To evaluate the presence of P. salmonis in gonads, gametes and reproductive fluids from

infected mothers (goal related with hypothesis 1).

2. To conduct monitoring for the offspring of mothers infected with P. salmonis up until the

fingerling phase (goal related with hypothesis 2).

Methodology for specific goal 1:

1. To verify the presence of bacteria in infected females, by means of immunofluorescence

(IFAT) and qPCR.

2. To obtain samples of ovaries, ova and ovarian fluid from the P. salmonis positive specimens.

3. To detect the presence of bacteria in the samples described above, through IFAT and qPCR.

4. To carry out microbiological cultures for the isolation of bacteria from the selected

samples.


Methodology for specific goal 2:

1. To verify the presence of bacteria in infected females through IFAT and qPCR.

2. To carry out the spawning process.

3. To fertilize the ova with semen from non-infected breeders.

4. To incubate the ova until hatching.

5. To maintain the culture until the first feeding in the fingerling phase.

6. To carry out regular sampling in each phase of the experiment, in order to monitor the

infective process.


CONCLUSIONS

“In the microbial systematics, the classification and identification of microorganisms is

based on the comparison of the physiological, biochemical and molecular characteristics of

the isolated strains.

Therefore, the microbial strains must be the reflection of the complete understanding and

interpretation of its genomic sequences, used to comprehend its biochemical reactions and

metabolic pathways, as well as to constitute reference organisms for research in microbial

ecology.

This means that the isolated and correctly characterized strains constitute the basis of not

only the microbial system, but also of the various applications that allow us to understand,

enhance and control them, where applicable (i.e. biotechnology, pharmacology, public

health).”

Microbial resource centers (MRCs) are units capable of protecting, maintaining and

distributing validated microbial strains, with defined genomic profiles and other information

associated with their functional in vivo expression.

They must allow for the deposit and distribution of strains that constitute the basis for a

comparative microbial taxonomy and its association with epidemiology, in the case of dealing

with organisms with pathogenic potential.

Apart from taxonomy, the deposited strains will facilitate the analysis and monitoring of

scientific studies associated with their variable functional behavior.

Only then will the MCR-Psal be able to generate a considerable added value through the

enrichment stages, enrichment selection, isolation, characterization, conservation and

long-term storage for the deposited strains, variants and isolates.

An important future challenge of the MCR-Psal will be to assure a higher fraction of strains


that are isolated in research laboratories around the world, providing expert knowledge and

essential culture and preservation skills.

For this, we have registered at the World Data Center for Microorganisms maintained by

the World Federation for Culture Collections (WFCC) at (http://www.wfcc.info/ccinfo/

collection/), under the acronym P-sal.

Hereinafter, a report will be presented of the results that contributed to a definition of an

operative platform, allowing for the establishment of an official, reliable and sustainable

P. salmonis strain collection, that contains the main variants/strains recovered from the

three salmonid species that are farmed in Chile, as a basis for the application of an efficient

sanitary management. The recovery of these variants/strains allows for the completion of

fundamental research regarding their eventual differences, for defining distinctive molecular

parameters associated with the biological behavior of the bacteria and with the development

of the disease, having a goal of designing strategies for its control in the field.

We were able to verify that the bacteria differs from other Gammaproteobacteria because

of the high and variable level of complexity of its genetic expression, which is absolutely

dependent on the farming conditions in which it finds itself. Despite having been classified

as a facultative bacteria, it essentially behaves as an obligate intercellular bacteria, given that

it presents a level of aggressiveness and virulence in vivo (varying for different isolates) that

is not compatible with the growth conditions tested in vitro, both in free cell systems and in

infections aimed at different cell lines. Its growth is scarce in these conditions in long time

periods, if any growth occurs at all, which hinders its cloning and the obtaining of its biomass

for a more finely-tuned genetic characterization.

In the context of the generation of a Strain Collection, this information is fundamental given

that one of the basic requirements for its constitution and maintenance is to be able to grow

the isolates, characterize them and maintain them over time. Fortunately, we have been

able to design procedures that have helped reactivate cryopreserved isolates for at least six

months after their freezing, limited to the evaluation of this report. However, we consider

that this is the ideal period, during the Strain Collection trial period, in which the cryo-frozen

isolates should be reactivated.


As was previously mentioned, each isolate has variable growth behavior in different media

and cell lines. We have been able to confirm this fact, given that of all of the different media

used, it is impossible to conclude that any one of these is better than the other. This is why we

recommend that each isolate is evaluated in at least two liquid media, two solid Agar media

and two cell lines.

Given the above, it is essential to establish a precise procedure that allows for the isolation

for the bacteria from naturally infected fish for its subsequent characterization and storage.

The conservation of tissues in different media becomes more and more complex as time

passes, and the capacity to recover viable bacteria with replicative capacities in the tested

artificial media is significantly reduced. Since we have been able to demonstrate that this

works with a relative degree of efficiency, we suggest recovering them directly from the

organs of recently sacrificed fish using a sterile swab stick and directly placing them in the

proposed solid media. Once the colonies are recovered, we proceed to the growing process in

liquid media, then to the verification of the infective capacity by exposure of the colony to

the proposed cell lines for its eventual growth, amplification, molecular characterization and

cryopreserved maintenance. To this effect, we have generated the first draft of an operative

manual that includes the adequate procedures for recovering the eventual variant of the

bacteria from naturally infected fish, for cloning in solid media (in order to obtain unique

colonies), for growing the colonies in liquid media and once they are molecularly categorized,

for cryopreserving them as new components of the Strain Collection.

Considering the above, it is moderately clear that the phenotypic expressions detected

under the best in vitro growing conditions are not conclusive for establishing a precise

categorization of the genetic quality and potential for each isolate. The only way to achieve

this is through the characterization of the P. salmonis isolates/variants/strains by means

of massive sequencing of all of their genetic material, and then realizing a meticulous

phylogenetic and metagenomics analysis with established bioinformatic procedures. We

have carried out this process in various steps. First of all, by cloning and sequencing PCR

products of the standard markers used for the phylogenetic characterization in bacteria: 16s

ribosomal RNA and the spacer sequence of the bacterial ribosomal operon known as ITS. We

carried out the analysis with the sequences of the existing isolate in our laboratories, as well


as of new variants that were recovered between December 2016 and May 2017, which allowed

us to obtain a wide variety of origins for the isolates. However, according to the information

explained in scientific literature, we mainly concentrated on two main groups: strain types

LF-89, isolated in Chile and EM-90, from Norway. However, there are certain variants that

tend to form an intermediate group; when using phylogenic markers in their analysis, it

was impossible to come to a conclusion regarding their pathogenic potential. Therefore, the

second step of the process has consisted in the selection of 20 isolates for sequencing and

metagenomic validation, putatively representing a wide range of diversity. In order to carry

out this process and their sequencing and interpretation, we had the support of one of the

most experienced analysis centers in Europe: the Leibniz Institute DSMZ – German Collection

of Microorganisms and Cell Cultures, led by Professor Dr. Jörg Overmann, the advisor of our

project. With the 20 closed genome sequences, still in process, we will be able to adequately

characterize the variability of the isolates. We recommend that the following procedure be

used once the Strain Collection is operational: recovery of the isolates directly from the fish,

growth and sequencing of the entire genome. The sequencing of the 20 isolates, representing

a wide variety of geographical origins and fish species (coming from at least two of the most

susceptible species to this bacteria, Salmo salar and Onchorynchus mykiss), will allow for

the foundation of the first outline of an epidemiological map, necessary for Sernapesca’s

decision-making process when confronted with outbreaks in the farming centers. Together

with this, the information that has been collected up to date, though still unavailable in Chile,

will help us define the first steps for consolidating management strategies not only for the

creation of an adequately characterized Strain Collection, but also for providing sustainability

and operative capacities over time. Given the above, and though it is not a product necessary

for this step of the project, we have decided to incorporate an advance of the type of analyses

that we will be able to carry out once we have the information of the complete and finished

genome in annex 2.

As we have concluded, the real pathogenic activity and the degrees of virulence that the

different bacterial isolates present must be evaluated in vivo. The in vitro evaluation

represents a valid approach; however it is inconclusive, constituting a robust interpretative

matrix system, when aided by the massive sequencing of its genomes and the interpretation of


its putative genes associated with the development of the disease. However, there is another

relevant factor associated with the behavior of this bacterium that needs to be clarified. This

is the fact that its total phenotypic expression, meaning the development of the pathology

that leads irretrievably to the death of affected fish, happens mainly in seawater rather than

freshwater. This is why it is crucial to analyze the behavior of the isolates in relation to how

the host responds with its capacity of controlling the disease. A pilot experiment in a wet

laboratory allowed for the confirmation of this fact: fish tested in both salinity conditions

presented different behaviors, where the pathogen developed only in seawater. A scalable

experiment that is currently in progress will allow us to correlate the mortality induction

with an evaluation of the host’s immunity markers, another element for our understanding

of the biological behavior of the bacteria. The recovered bacteria from the test in both salinity

conditions will be evaluated in vitro for their infective potential.

A complementary element to the structuring of the Strain Collection, relevant for the

understanding of the bacteria’s biological behavior in its natural environment, is to know

how it is maintained and transferred. We must not forget that salmon farming in Chile is an

activity that depends on the introduction of non-native fish into certain environments, in

order to generate confined and restricted growth. Along with the introduction of the fish,

came this pathogenic bacterium, which requires the understanding of its maintenance and

evolution. To this effect, we have considered whether there are organisms in the water column

that are being used by bacteria as potential biological or mechanical vectors or as intermediate

hosts, associated with the fish’s confinement and growth in cages. A fragmented analysis of

the water column from the Neuston to the sediment under cages has allowed us to detect

the existence of unicellular organisms similar to microalgae in terms of the sequencing of

their phylogenetic markers. Therefore, we have designed an association experiment for this

bacteria and a laboratory microalga in order to determine whether the bacteria is capable of

infecting it, expressing itself and/or reproducing in the microalga.


Lastly, regarding the mechanisms involved in the vertical transmission of the disease, we

can confirm that our research in bibliographic data has shown that this is not the preferred

transmission channel for aquatic pathogens, with the exception of BKD. However, despite

the fact that it is an improbable and complicated alternative, it is still a possible transmission

channel for P. salmonis, and this is why we will continue our efforts of clarifying this

transmission channel.

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