# Population structure of the fish pathogen Flavobacterium psychrophilum at whole-country and model river levels in Japan

Erina Fujiwara-Nagata, Céline Chantry-Darmon, Jean-François Bernardet, Mitsuru Eguchi, Éric Duchaud, Pierre P. Nicolas

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## To cite this version:

Erina Fujiwara-Nagata, Céline Chantry-Darmon, Jean-François Bernardet, Mitsuru Eguchi, Éric Duchaud, et al.. Population structure of the fish pathogen Flavobacterium psychrophilum at whole-country and model river levels in Japan. Veterinary Research, 2013, 44, pp.1-9. 10.1186/1297-9716-44-34. hal-02651630

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RESEARCH

Open Access

# Population structure of the fish pathogen *Flavobacterium psychrophilum* at whole-country and model river levels in Japan

Erina Fujiwara-Nagata<fnref n="1" />,<fnref n="2" />*, Céline Chantry-Darmon<fnref n="2" />,³, Jean-François Bernardet<fnref n="2" />, Mitsuru Eguchi<fnref n="1" />, Eric Duchaud<fnref n="2" />† and Pierre Nicolas³†

## Abstract

The bacterium *Flavobacterium psychrophilum* is a serious problem for salmonid farming worldwide. This study investigates by multilocus sequence typing (MLST) the population structure of this pathogen in Japan where it is also a major concern for ayu, a popular game fish related to salmoniforms. A total of 34 isolates collected across the country and 80 isolates sampled in a single model river by electrofishing were genotyped. The data accounting for 15 fish species allowed identifying 35 distinct sequence types (ST) in Japan. These ST are distinct from those reported elsewhere, except for some ST found in rainbow trout and coho salmon, two fish that have been the subject of intensive international trade. The pattern of polymorphism is, however, strikingly similar across geographical scales (model river, Japan, world) in terms of the fraction of molecular variance linked to the fish host (~50%) and of pairwise nucleotide diversity between ST (~5 Kbp⁻<fnref n="1" />). These observations go against the hypothesis of a recent introduction of *F. psychrophilum* in Japan. Two findings were made that are important for disease control: 1) at least two independent *F. psychrophilum* lineages infect ayu and 2) co-infections of the same individual fish by different strains occur.

## Introduction

Diseases are an important problem in fish farming and control of bacterial pathogens relies mostly on repeated antibiotic treatments incompatible with the development of sustainable aquaculture [1]. The fish-pathogenic bacterium *Flavobacterium psychrophilum* is the causative agent of the “bacterial cold water disease” (BCWD) and the “rainbow trout fry syndrome”, two salmonid diseases that cause considerable losses in rainbow trout (*Oncorhynchus mykiss*) and coho salmon (*Oncorhynchus kisutch*) farming industries worldwide [2-5]. In Japan, *Flavobacterium psychrophilum* was first isolated in 1987 from cultured ayu (*Plecoglossus altivelis altivelis*), a fish related to salmoniforms [6]. The status of *F. psychrophilum* in the country before the development of the fish farming industry and possible importation from foreign sources is unclear [7-9] but it is currently recognized as a serious

problem, in particular for ayu [10]. Indeed, to increase the stocks of this very popular game fish, juveniles hatched from artificially fertilized eggs or captured in coastal areas are released into rivers by fish farming companies and experimental stations located in each prefecture. The bacterium is then probably disseminated with ayu juveniles. Infections are now repeatedly reported not only in fish farms but also in natural environments [6,11].

Many species beyond the ayu and farmed rainbow trout and coho salmon have been reported to harbor *F. psychrophilum* in Japan. These include migrating salmonids whose geographical ranges extend throughout the north-Pacific and more local species and subspecies. The actual host range of *F. psychrophilum* is, however, not limited to salmonids [12], although the infection is apparently less severe in non-salmonids [13]. In Japan, *F. psychrophilum* has been reported on a number of local non-salmonid wild fish species [14-16].

Understanding the degree and determinants of host specificity of *F. psychrophilum* strains as well as their routes of dissemination would help setting rational

<footnote>* Correspondence: erina_nagata@nara.kindai.ac.jp</footnote>

<footnote>†Equal contributors</footnote>

<footnote n="1">¹Department of Fisheries, Kinki University, Nara, Japan</footnote>

<footnote n="2">²INRA, Virologie et Immunologie Moléculaires UR892, Jouy-en-Josas, France</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
practices to limit the spreading of the disease. Correla-
tions between bacterial genotype and host fish have been
observed in several studies on field isolates [14,17-20]
and we recently reported that ayu could be experimen-
tally infected in bath infection challenges by isolates col-
lected from ayu but not from coho salmon or rainbow
trout [14]. However mimicking the conditions of natural
infections is generally difficult for *F. psychrophilum*
[21-24] and field studies that would provide a deeper
knowledge of the distribution of the genotypes in terms
of geographical area and host fish species are essential.

Following the publication of the complete genome
sequence of a *F. psychrophilum* strain [25], a first Multi-
locus sequence typing (MLST) analysis was conducted
on a set of 50 isolates collected from 11 different fish
species in 12 different countries over the world [17].
This initial study identified 33 different sequence types
(ST), some of which could be grouped into clonal com-
plexes (CC) associated with particular fish species, espe-
cially rainbow trout and coho salmon. The results also
revealed remarkably high rates of homologous recom-
bination and limited nucleotide diversity. However, the
small number of isolates from each particular geograph-
ical origin did not allow the description of population
structure at a regional scale. A more recent analysis of
66 additional isolates from farmed rainbow trout in
France [26] concluded to the predominance of a single
clonal complex. In the present study we investigated the
genetic diversity of *F. psychrophilum* in Japan using a
higher number of isolates than for any other region and
taking into account the diversity of fish hosts. We also
compare the patterns of polymorphism at the world,
country and single model river levels.

**Materials and methods**

Field sampling of *F. psychrophilum* isolates

A first set of Japanese *F. psychrophilum* isolates was
contributed by correspondents all over Japan (listed in
Additional file 1). A second set of isolates was collected
in the downstream area of the ~9.5 km long Chinai River
near (~0.3 km) its mouth into Lake Biwa, the largest lake
in Japan. With an authorization of the Shiga prefecture,
fish were caught by electrofishing, kept at low temperature
into tanks, quickly transported to our laboratory and
euthanized by severing their spinal cord. Gill samples were
cut into pieces of ~25 mm² and suspended in 1 mL of
phosphate buffered saline (PBS). The tubes were vortexed
during 30 s at maximum speed and the suspensions were
further diluted in PBS as to obtain well-separated colonies
on modified cytophaga (MCYT) agar [27]. Samples from
internal organs and skin lesions were collected using a
sterile loop and streaked directly onto MCYT plates.
Water samples (containing 2000 ~ 3000 cfu mL⁻¹) were

diluted to 10⁻⁴ in PBS prior to being spread onto MCYT
plates. Caddisfly cases were vigorously vortexed in 1.5 mL
of PBS and the suspensions were diluted and spread onto
MCYT plates. All plates were incubated at 15°C for 4 days.
Yellow colonies were selected for identification as *F. psychrophilum* based on the PCR amplification of the 16S
rRNA and *gyrB* genes [18,28,29] according to the guide-
lines of the council for the control of bacterial cold water
disease of ayu (Japanese Ministry of Agriculture, Forestry
and Fisheries). Each isolate received a unique identifier
containing the date of sampling encoded in the “yymmdd”
format.

Multi locus sequence typing

The seven target loci as well as the PCR and sequencing
protocols are listed in Additional file 2. The sequences
were assembled using Phred/Phrap/Consed [30], verified
manually to ensure high quality, and deposited in
Genbank (GenBank:KC203886-KC204683). In keeping
with MLST standards [31,32], arbitrary numbers served
for unambiguous identification of the AT (particular al-
leles at particular loci) and ST (unique combinations of
AT at the seven loci). To facilitate large scale studies the
following changes were made compared to the initial
study [17]: PCR amplification was performed with an
optimized touchdown protocol; 5' extensions were added
to the PCR primers to allow sequencing of the 7 loci
with the same pair of primers; and AT attribution was
based on slightly shortened sequences. The data, inclu-
ding the genotypes from [17,26], are made available in a
new version of the *F. psychrophilum* MLST database
[33] which uses the BIGSdb system [34].

A tree depicting AT-sharing between ST was built by
hierarchical clustering with R; the function "hclust"
using the single link aggregation method and drawn with
the R package "ape". The eBURSTv3 software with de-
fault settings [35] was used to obtain a "population snap-
shot" highlighting the relationships between closely
related ST. Clonal complexes (CC) were defined using
the criterion of single locus variant (SLV) connections
between ST. Analyses of nucleotide diversity, including
analysis of molecular variance [36], were coded in R.
Nucleotide diversity was computed on pairwise compari-
sons between unique ST rather than between isolates to
minimize the impact of sampling biases; as a conse-
quence, the values were slightly higher than those ini-
tially reported in Nicolas et al. [17].

**Results and discussion**

Sampling of *F. psychrophilum* diversity at whole-country
and model river levels

We analyzed bacterial isolates representative of two
different geographical levels. In addition to 6 MLST
profiles already available [17], we genotyped a first set of
34 *F. psychrophilum* isolates collected between 1993 and
2005 from a variety of locations all over Japan to ac-
count for the diversity at the country level. Our second
set consisted of 80 isolates obtained for this study be-
tween August 2005 and December 2006 from a single
model river in which fish were captured irrespective of
their disease status to minimize sampling biases. This
strategy allowed retrieving isolates from 6 fish species, as
well as 4 isolates from water samples and 1 from the
case of a caddisfly (order *Trichoptera*). This raises the
number of MLST profiles available to study the
*F. psychrophilum* population structure in Japan to 120
and the number of sampled host fish species to 15.
Table 1 provides a detailed summary of the data. These
could be compared to 110 other MLST profiles from the
rest of the world [17,26].

Population structure at the country level

Our MLST data reveal a high genetic diversity of
*F. psychrophilum* in Japan. The total number of different
ST in the Japanese isolate collection amounts to 35,
among which 32 are only reported in Japan (Figure 1).
At the nucleotide level, these 35 ST harbor a pairwise
nucleotide diversity of 5.4 Kbp⁻¹ comparable to the 5.6
Kbp⁻¹ found in the 230 isolates representative of world-
wide diversity. This indicates that the structure of the
*F. psychrophilum* population is at most only marginally
linked to geographical origin. Indeed, in our data, as
much as 88% of the nucleotide sites that are poly-
morphic in Europe are also polymorphic in Japan.

Many of the ST that were sampled several times are
associated with the defined host fish (Figures 1 and 2).
The ST of most Japanese isolates (76%) could indeed be
classified into six CC and the ST from a same CC also
tended to infect similar host fish. Each CC is named
after a representative ST. These six CC are the following:
CC-ST52 (59 isolates) from ayu; CC-ST48 (13 isolates)
from ayu; CC-ST56 (12 isolates) from ayu and cyprinids;
CC-ST2 (3 isolates) from rainbow trout; CC-ST9 (2 iso-
lates) from coho salmon; and CC-ST54 (2 isolates) from
masou salmon. Of note, CC-ST48 and CC-ST56 that
both infect ayu are connected by a double locus variant
(DLV) link and therefore probably share a recent com-
mon ancestor, even though here they are distinguished
using the stringent operational definition of CC based
on the SLV links.

Analysis of the molecular variance confirmed that
the association between genotype and the host fish in
Japan was highly statistically significant and showed
that it explains as much as 44.2% of the genetic vari-
ance. This level was slightly lower but close to the
51.3% reported at the worldwide level by Nicolas et al.

[17] and to the 48.3% computed here on the new set
of 230 isolates.

At least two separate lineages (CC-ST52 and CC-ST48/
CC-ST56) infect this fish species, a fact that is important
for the future treatment and prevention of BCWD in ayu.
Strains in these three CC showed various levels of viru-
lence against ayu in bath infection challenges and strains
from CC-ST48/CC-ST56 generally seem more virulent
than those from CC-ST52 [14]. Of note, CC-ST56 also
infects cyprinids in Japan and [14] reported that ST-51
isolates from cyprinids cause mortality in ayu by bath
infection challenge, providing experimental support
for the possibility of transfer between carp and ayu in
CC-ST56.

Footprints and possible mechanisms of transcontinental
dissemination

The absence of clear genetic signal reflecting the geog-
raphy indicates mixing between *F. psychrophilum* popula-
tions located on different continents. Such a deep effect
on the whole population probably involved recurrent nat-
ural dissemination before the development of commercial
exchanges. This may seem surprising for a fish-pathogenic
bacterium found in freshwater that one can believe limi-
ted by watersheds. However, long-distance dissemination
events do not need to be frequent to have a substantial
impact on genetic structure: a few migrants per generation
in the entire population have dramatic homogenizing
consequences in idealized models [37,38]. Potential me-
chanisms include fish and bird migrations but airborne
dissemination may be another possibility. Indeed, *F.
psychrophilum* is able to survive in pure water [39] and
freshwater bacteria -including *Flavobacteriaceae-* were
recently sampled in the upper troposphere [40]. Alterna-
tively, genetic mixing can involve a succession of more
frequent small-distance steps. The DLV link between
ST44 sampled in Japan from *H. nipponensis* and ST104
sampled in France from rainbow trout might be a foot-
print of natural dissemination. Both ST have been sam-
pled only once and are not connected to other genotypes,
which suggests that the ST44/ST104 lineage infects farmed
fish only sporadically and should thus not be prone to
dissemination by human activities.

In sharp contrast, the three ST also reported in other
countries (ST9, ST10 and ST13) are associated to rain-
bow trout and coho salmon. These are two fish species
with wide geographical distribution and whose eggs and
broodfish have been imported to Japan (vertical trans-
mission from parent to offspring via eggs is highly
suspected [41]). Two other ST that we found in Japan
are connected by SLV links to ST found abroad: ST17,
sampled twice from rainbow trout, is linked to the main
rainbow trout clonal complex (CC-ST2); and CC-ST56,
infecting ayu and cyprinids in Japan, is linked to ST14
<table><caption>Table 1 MLST profiles and background information for the 120 *F. psychrophilum* isolates from Japan</caption><thead><tr><th>Isolate<sup>a</sup></th><th>ATs<sup>b</sup></th><th>ST<sup>c</sup></th><th>Prefecture<sup>d</sup></th><th>Host<sup>e</sup></th><th>Tissue</th><th>Year</th></tr></thead><tbody><tr><td>FPC 837*</td><td>5, 4, 4, 4, 2, 4, 4</td><td>ST5</td><td>Tokushima</td><td>P. altivelis (ayu)</td><td>Kidney</td><td>1987</td></tr><tr><td>FPC 840*</td><td>5, 4, 4, 4, 2, 4, 4</td><td>ST5</td><td>Tokushima</td><td></td><td>Kidney</td><td>1987</td></tr><tr><td>PH9351</td><td>19,24,19, 5, 8, 1, 8</td><td>ST45</td><td>Hiroshima</td><td></td><td>Ovary</td><td>1993</td></tr><tr><td>CS-1</td><td>19,24,19, 5, 8, 1, 8</td><td>ST45</td><td>Gifu</td><td></td><td>Kidney</td><td>1995</td></tr><tr><td>96-4</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52</td><td>Gifu</td><td></td><td>Kidney</td><td>1996</td></tr><tr><td>PH-0003</td><td>19,24,19, 5, 8, 1, 8</td><td>ST45</td><td>Hiroshima</td><td></td><td>Kidney</td><td>2000</td></tr><tr><td>SG011227</td><td>5,24, 2, 6, 8, 1, 8</td><td>ST56</td><td>Shiga</td><td></td><td>Kidney</td><td>2001</td></tr><tr><td>PH-0209</td><td>19,24,19, 6, 8, 1, 8</td><td>ST48</td><td>Hiroshima</td><td></td><td>Kidney</td><td>2002</td></tr><tr><td>AK-0527</td><td>19,19,19, 2,13,29,28</td><td>ST53</td><td>Kyoto</td><td></td><td>Lower jaw</td><td>2005</td></tr><tr><td>AK-0536</td><td>19,19,19, 2,13,29,28</td><td>ST53</td><td>Kyoto</td><td></td><td>Lower jaw</td><td>2005</td></tr><tr><td>AK-0531</td><td>19,19,19, 2,13,29,28</td><td>ST53</td><td>Kyoto</td><td></td><td>Kidney</td><td>2005</td></tr><tr><td>AK-05137</td><td>19,19,19, 2,13,29,28</td><td>ST53</td><td>Kyoto</td><td></td><td>Skin lesion</td><td>2005</td></tr><tr><td>KU050822-1</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52</td><td>Shiga (MR)</td><td></td><td>Skin lesion</td><td>2005</td></tr><tr><td>KU050822-3 ~ 4</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(2)</td><td>Shiga (MR)</td><td></td><td>Skin lesion</td><td>2005</td></tr><tr><td>KU051024-5</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52</td><td>Shiga (MR)</td><td></td><td>Gill</td><td>2005</td></tr><tr><td>KU051024-6</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52</td><td>Shiga (MR)</td><td></td><td>Coelomic fluid</td><td>2005</td></tr><tr><td>KU051024-7</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52</td><td>Shiga (MR)</td><td></td><td>Coelomic fluid</td><td>2005</td></tr><tr><td>KU051024-8 ~ 11</td><td>5,24, 2, 6,10, 1, 8</td><td>ST65(4)</td><td>Shiga (MR)</td><td></td><td>Egg</td><td>2005</td></tr><tr><td>KU051024-12 ~ 13</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(2)</td><td>Shiga (MR)</td><td></td><td>Egg</td><td>2005</td></tr><tr><td>KU051024-14 ~ 17</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(4)</td><td>Shiga (MR)</td><td></td><td>Egg</td><td>2005</td></tr><tr><td>KU060427-1</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52</td><td>Shiga (MR)</td><td></td><td>Gill</td><td>2006</td></tr><tr><td>KU060626-1 ~ 3</td><td>19,24,19, 6, 8, 1, 8</td><td>ST48(1)</td><td>Shiga (MR)</td><td></td><td>Kidney</td><td>2006</td></tr><tr><td></td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(2)</td><td>-</td><td></td><td>-</td><td>-</td></tr><tr><td>KU060626-4 ~ 6</td><td>5,24, 2, 6, 8, 1,27</td><td>ST49(3)</td><td>Shiga (MR)</td><td></td><td>Kidney</td><td>2006</td></tr><tr><td>KU060626-7 ~ 9</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(3)</td><td>Shiga (MR)</td><td></td><td>Kidney</td><td>2006</td></tr><tr><td>KU060626-10 ~ 12</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(3)</td><td>Shiga (MR)</td><td></td><td>Kidney</td><td>2006</td></tr><tr><td>KU060626-13 ~ 15</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(3)</td><td>Shiga (MR)</td><td></td><td>Kidney</td><td>2006</td></tr><tr><td>KU060626-16 ~ 18</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(3)</td><td>Shiga (MR)</td><td></td><td>Kidney</td><td>2006</td></tr><tr><td>KU060626-19 ~ 21</td><td>19,24,19, 6, 8, 1, 8</td><td>ST48(2)</td><td>Shiga (MR)</td><td></td><td>Kidney</td><td>2006</td></tr><tr><td></td><td>5,24, 2, 5, 8, 1,35</td><td>ST67(1)</td><td>-</td><td></td><td>-</td><td>-</td></tr><tr><td>KU060626-22 ~ 24</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(3)</td><td>Shiga (MR)</td><td></td><td>Kidney</td><td>2006</td></tr><tr><td>KU060626-25 ~ 27</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(3)</td><td>Shiga (MR)</td><td></td><td>Kidney</td><td>2006</td></tr><tr><td>KU060626-28 ~ 36</td><td>19,24,19, 6, 8, 1, 8</td><td>ST48(1)</td><td>Shiga (MR)</td><td></td><td>Kidney</td><td>2006</td></tr><tr><td></td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(7)</td><td>-</td><td></td><td>-</td><td>-</td></tr><tr><td>KU060626-37 ~ 39</td><td>19,24,19, 6, 8, 1, 8</td><td>ST48(3)</td><td>Shiga (MR)</td><td></td><td>Kidney</td><td>2006</td></tr><tr><td>KU060626-40 ~ 42</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(3)</td><td>Shiga (MR)</td><td></td><td>Kidney</td><td>2006</td></tr><tr><td>KU060626-43 ~ 46</td><td>19,24,19, 6, 8, 1, 8</td><td>ST48(1)</td><td>Shiga (MR)</td><td></td><td>Kidney</td><td>2006</td></tr><tr><td></td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(3)</td><td>-</td><td></td><td>-</td><td>-</td></tr><tr><td>KU060626-57</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52</td><td>Shiga (MR)</td><td></td><td>Skin lesion</td><td>2006</td></tr><tr><td>KU060626-59</td><td>19,24,19, 6, 8, 1, 8</td><td>ST48</td><td>Shiga (MR)</td><td></td><td>Skin lesion</td><td>2006</td></tr><tr><td>KU060920-5 ~ 6</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(2)</td><td>Shiga (MR)</td><td></td><td>Gill</td><td>2006</td></tr><tr><td>KU060920-7 ~ 8</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52(2)</td><td>Shiga (MR)</td><td></td><td>Gill</td><td>2006</td></tr><tr><td>KU060920-19</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52</td><td>Shiga (MR)</td><td></td><td>Milt</td><td>2006</td></tr></tbody></table>
<table><caption>Table 1 MLST profiles and background information for the 120 *F. psychrophilum* isolates from Japan (Continued)</caption><tr><td>OH-0224</td><td>2,30,20, 2,22,22,23</td><td>ST54</td><td>Hiroshima</td><td>O. masou subsp. (resp. yamame, amago x2, and biwamasu)</td><td>Gill</td><td>2003</td></tr><tr><td>96-1</td><td>17,26,18, 9,19,24,24</td><td>ST43</td><td>Gifu</td><td></td><td>Kidney</td><td>1996</td></tr><tr><td>OH-0519</td><td>2,30,20, 2,23,22,23</td><td>ST55</td><td>Hiroshima</td><td></td><td>Kidney</td><td>2005</td></tr><tr><td>SG030207</td><td>15,24,10, 3, 2,12,11</td><td>ST40</td><td>Shiga</td><td></td><td>Kidney</td><td>2003</td></tr><tr><td>FPC 813*</td><td>2, 8, 2, 2, 2,10, 2</td><td>ST17</td><td>Tokyo</td><td>O. mykiss (rainbow trout)</td><td>Kidney</td><td>1992</td></tr><tr><td>FPC 814*</td><td>2, 8, 2, 2, 2,10, 2</td><td>ST17</td><td>Tokyo</td><td></td><td>Kidney</td><td>1992</td></tr><tr><td>CS-3</td><td>14,23, 4,10, 6,22, 2</td><td>ST39</td><td>Gifu</td><td></td><td>Kidney</td><td>1995</td></tr><tr><td>SG950607</td><td>2, 8, 2, 2, 2, 2, 2</td><td>ST10</td><td>Shiga</td><td></td><td>Kidney</td><td>1995</td></tr><tr><td>SG010808</td><td>2,29,15,12,17,27,26</td><td>ST47</td><td>Shiga</td><td></td><td>Kidney</td><td>2001</td></tr><tr><td>OH-0203</td><td>2, 8, 2, 2, 2, 2, 2</td><td>ST10</td><td>Hiroshima</td><td></td><td>Kidney</td><td>2002</td></tr><tr><td>0312</td><td>7,28, 4, 5,21,28,21</td><td>ST50</td><td>Yamanashi</td><td></td><td>Kidney</td><td>2003</td></tr><tr><td>y-2</td><td>2, 8, 2, 2, 2, 2, 2</td><td>ST10</td><td>Iwate</td><td></td><td>Kidney</td><td>2005</td></tr><tr><td>y-3</td><td>4,25, 4, 5,18,23,23</td><td>ST42</td><td>Iwate</td><td></td><td>Kidney</td><td>2005</td></tr><tr><td>FPC 830*</td><td>4, 7, 6, 5, 6, 8, 4</td><td>ST13</td><td>Miyagi</td><td>O. kisutch (coho salmon)</td><td>Kidney</td><td>1990</td></tr><tr><td>FPC 831*</td><td>11,18, 7, 5,14,18,17</td><td>ST30</td><td>Iwate</td><td></td><td>Peduncle</td><td>1990</td></tr><tr><td>FPM960724</td><td>11,18, 7, 5,14,18,17</td><td>ST30</td><td>Miyagi</td><td></td><td>not available</td><td>1996</td></tr><tr><td>FPM960726</td><td>4, 7, 6, 5, 6, 5, 4</td><td>ST9</td><td>Miyagi</td><td></td><td>Kidney</td><td>1996</td></tr><tr><td>x-2</td><td>11,18, 7, 5,14,18,17</td><td>ST30</td><td>Iwate</td><td></td><td>Kidney</td><td>2005</td></tr><tr><td>SG020617</td><td>16, 4,17, 5,10,12,22</td><td>ST41</td><td>Shiga</td><td>S. leuc. (iwana)</td><td>Kidney</td><td>2002</td></tr><tr><td>SG980216</td><td>18,27,10, 7,20,25,25</td><td>ST44</td><td>Shiga</td><td>H. nipponensis (wakasagi)</td><td>Egg</td><td>1998</td></tr><tr><td>SG010619</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52</td><td>Shiga</td><td></td><td>Kidney</td><td>2001</td></tr><tr><td>SG040302</td><td>20,31,19, 2,23,30,28</td><td>ST57</td><td>Shiga</td><td></td><td>Kidney</td><td>2004</td></tr><tr><td>GM2127</td><td>4,28, 8,11, 1,26, 4</td><td>ST46</td><td>Gunma</td><td>T. hakonensis (ugui)</td><td>Skin lesion</td><td>1999</td></tr><tr><td>CH-9401</td><td>5,24, 8, 6, 8, 1, 8</td><td>ST51</td><td>Hiroshima</td><td>C. carpio (koi)</td><td>Skin lesion</td><td>1994</td></tr><tr><td>KU061226-2 ~ 3</td><td>24,21,15, 1, 8,34,33</td><td>ST63(2)</td><td>Shiga (MR)</td><td>Cyprinid fish</td><td>Gill</td><td>2006</td></tr><tr><td>CH-0411</td><td>5,24, 8, 6, 8, 1, 8</td><td>ST51</td><td>Hiroshima</td><td>C. carassius (funa)</td><td>Kidney</td><td>2004</td></tr><tr><td>KU061226-1</td><td>25,35,10, 2,16, 1, 4</td><td>ST64</td><td>Shiga (MR)</td><td>P. jouyi (takahaya)</td><td>Gill</td><td>2006</td></tr><tr><td>PH-9348</td><td>21,32,10, 7,24,31,29</td><td>ST58</td><td>Hiroshima</td><td>Z. platypus (oikawa)</td><td>Kidney</td><td>1993</td></tr><tr><td>ZH-0001</td><td>5,24, 8, 6, 8, 1, 8</td><td>ST51</td><td>Hiroshima</td><td></td><td>Kidney</td><td>2000</td></tr><tr><td>KU051024-4</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52</td><td>Shiga (MR)</td><td>T. bre. (numachichibu)</td><td>Gill</td><td>2005</td></tr><tr><td>KU051128-1</td><td>1,15,16, 5,19,35,34</td><td>ST66</td><td>Shiga (MR)</td><td>Gymnogobius sp.</td><td>Gill</td><td>2005</td></tr><tr><td>KU051128-4</td><td>23,29,11,13,13, 5,1</td><td>ST60</td><td>Shiga (MR)</td><td>L. rei. (sunayatsume)</td><td>Gill</td><td>2005</td></tr><tr><td>KU051128-8</td><td>23,29,11,13,13, 5,1</td><td>ST60</td><td>Shiga (MR)</td><td>River water</td><td>not applicable</td><td>2005</td></tr><tr><td>KU051128-10</td><td>22,31, 4,10,19,32,3</td><td>ST59</td><td>Shiga (MR)</td><td></td><td>not applicable</td><td>2005</td></tr><tr><td>KU060920-2</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52</td><td>Shiga (MR)</td><td></td><td>not applicable</td><td>2006</td></tr><tr><td>KU060920-4</td><td>5,24, 4, 4, 2, 4, 4</td><td>ST52</td><td>Shiga</td><td></td><td>not applicable</td><td>2006</td></tr><tr><td>KU061128-1</td><td>5, 34, 8, 1, 26, 26, 32</td><td>ST62</td><td>Shiga (MR)</td><td></td><td>not applicable</td><td>2006</td></tr><tr><td>KU051128-6</td><td>8, 33, 8, 1, 25, 33, 31</td><td>ST61</td><td>Shiga (MR)</td><td>Case of caddisfly</td><td>not applicable</td><td>2005</td></tr></table>

<footnote>a Isolate identifier. Isolates are ordered by host fish species and then by sampling year; * indicates isolates already included in Nicolas et al. [17]. When several isolates have been collected from the same individual fish their identifiers are reported in a condensed format (i.e. KU060626-40 ~ 42 stands for KU060626-40, KU060626-41 and KU060626-42).</footnote>

<footnote>b Allele types (AT) for the seven loci ordered trpB, gyrB, dnaK, fumC, murG, and atpA.</footnote>

<footnote>c Sequence type (ST). When several isolates were collected from the same individual fish the number in parentheses indicates the frequency of each ST. If different ST were found in the same fish, each ST is reported on a separate line but information on prefecture, tissue and year are not repeated (replaced by -).</footnote>

<footnote>d Geographical origin of the isolate in Japan. (MR) indicates an isolate from the model river in the Shiga prefecture.</footnote>

<footnote>e The host fish species or environmental sample source. The common name used in Japan is given in parentheses. Genus and sometimes species names have been abbreviated in this column. Full species names: *Plecoglossus altivelis*, *Oncorhynchus masou*, *Oncorhynchus mykiss*, *Oncorhynchus kisutch*, *Salvelinus leucomaenis*, *Hypomesus nipponensis*, *Trybolodon hakonensis*, *Cyprinus carpio*, *Carassius carassius*, *Phoxinus jouyi*, *Zacco platypus*, *Tridentiger brevispinis*, and *Lethenteron reissneri*.</footnote>
<figure><img src="image_4.png" /></figure>

sampled from a carp in Germany. In both cases, com-
mercial exchanges may be invoked to explain these SLV
links between Japanese and European isolates. Namely,
carps have been traded between Japan and Europe, and
rainbow trout have been imported from North America
to both Japan and Europe. Of note, the diversity of
CC-ST48/CC-ST56 found in Japan is a strong indication
that dissemination of this lineage occurred from Japan
to Europe rather than the contrary; altogether we found
no indication suggesting that strains infecting ayu might
have been recently imported from foreign sources.

Population structure in the model river

Extensive sampling of a model river unveils an unantici-
pated level of diversity in a restricted geographical area.
We found 12 different ST in the model river and a gene-
ral pattern of polymorphism surprisingly similar to that
of the whole country and worldwide levels. Namely, the
average pairwise nucleotide difference between the ST
was 4.8 Kbp⁻¹ and fish host accounted for 51.2% of the
molecular variance.

The isolates from the model river include representa-
tives of the three clonal complexes reported in ayu. The
<figure><img src="image_5.png" /></figure>

majority of ayu isolates belonged to ST52 and this ST was
observed over several seasons (i.e., August 2005, October
2005, April 2006, June 2006, and September 2006). Simul-
taneously, strains displaying ST52 were also isolated from
numachichibu and a water sample suggesting contami-
nation by ayu. A geographical population structure could
explain the dominance of CC-ST52 in the model river not
seen at the country level. However, at least two other fac-
tors may contribute to this difference: a temporal evolu-
tion of relative frequencies, since most isolates sampled at
the country level were collected years before the sampling
of the model river; and the fish sampling strategy, since
sampling in the model river was performed irrespectively
of disease symptoms and CC-ST52 seems less virulent
[14]. Seven ST (ST59, ST60, ST61, ST62, ST63, ST64,
and ST66) not connected to those found in ayu were
retrieved from river water, a case of caddisfly, and seve-
ral local fish. These strains were isolated in November
2005, November 2006 and December 2006, when ayu
are absent from the river due to their one-year life cycle
ending after spawning in the autumn. Six of these strains

(no data available on ST66) showed no pathogenicity
against ayu [14]. These ST may have gone unnoticed
if other fish species and the environment had not been
sampled.

Importantly, co-infection of the same individual fish
by different lineages was detected four times in ayu
(Table 1). This observation could have a practical inter-
est in the context of disease prevention or treatment but
could also have important evolutionary implications. In
particular, co-infection might promote evolution to-
wards higher virulence [36], in particular if distinct
transmission modes –horizontal and vertical- are in-
volved [42]. The local diversity of *F. psychrophilum*
genotypes detected in the model river, which goes down
to the level of the individual host fish, also certainly
contributes to explain the very high recombination rate
detected in the species on the basis of MLST data
[17,43]. Indeed, a greater local diversity provides more
opportunities to exchange alleles and therefore directly
increases the apparent recombination rate and the
actual genetic mixing.
## Additional files

**Additional file 1: Isolates representative of diversity at the country level.** Institutions that provided these isolates and previous publications on these isolates are listed [11,14,16,44-47].

**Additional file 2: MLST scheme.** PCR and sequencing primers for the seven loci as well as the corresponding experimental protocols are provided.

### Abbreviations

BCWD: Bacterial cold water disease; PCR: Polymerase chain reaction;

PBS: Phosphate buffered saline; MLST: Multilocus sequence typing; CC: Clonal

complex; ST: Sequence type; AT: Allele type; SLV: Single locus variant;

DLV: Double locus variant.

### Competing interests

The authors declare that they have no competing interests.

### Authors' contributions

EFN performed fish sampling in the model river, bacterial isolation, and contributed to sample processing, data analysis and writing of the manuscript. CCD contributed to the development and optimization of the MLST scheme and to sample processing. JFB and ME contributed to data analysis and presentation of the results. ED initiated the project and contributed to data analysis. PN analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.

### Acknowledgements

We are grateful to the organizations listed in Additional file 1 for providing *F. psychrophilum* strains. We also wish to thank Dr K. Sugahara, J. Ikeda, and A. Fukuda for help with field sampling and technical assistance and Dr Keith Jolley at University of Oxford for hosting the *F. psychrophilum* MLST database. This work received the support of a Grant-in-Aid for Young Scientists, KAKENHI (B) (18780153) and a Grant-in-Aid from the Global Center of Excellence (COE) Program, Japan. EFN was also supported by Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists and PN, CCD, JFB and ED by ANR (ANR-07-GMGE-004 Flavophylogenomics) and EU EMIDA ERA-NET (ANR 2010-EMID-006-01 Pathofish).

### Author details

Received: 28 November 2012 Accepted: 25 April 2013

Published: 17 May 2013

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doi:10.1186/1297-9716-44-34

**Cite this article as:** Fujiwara-Nagata *et al*: Population structure of the fish pathogen *Flavobacterium psychrophilum* at whole-country and model river levels in Japan. *Veterinary Research* 2013 **44**:34.

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