Serological and molecular typing of Tenacibaculum maritimum from New Zealand farmed salmon, Oncorhynchus tshawytscha

Abstract

Tenacibaculum maritimum is a cosmopolitan bacterial pathogen with the potential to cause significant losses in a broad range of farmed and wild marine fish species. This study investigated the antigenic diversity of T. maritimum isolated in culture from farmed Chinook (king) salmon (Oncorhynchus tshawytscha) from the Marlborough Sounds in New Zealand. A total of 36 isolates were examined using antibody serotyping and rapid molecular serotyping via multiplex PCR (mPCR) targeting genes encoding O-antigen biosynthesis enzymes. Serological analysis using three different polyclonal antisera developed against T. maritimum isolated from farmed Atlantic salmon (Salmo salar) in Tasmania, Australia revealed that there are three putative serotypes of T. maritimum that occur in New Zealand. The predominant serotype was defined by a positive reaction to all three Tasmanian antisera (antisera A, B and C), designated as serotype ABC. This serotype was found at all nine farm locations tested and represented 81% of all isolates examined. The same library of isolates was evaluated by mPCR serotyping and found three O-AGC types among tested isolates. O-AGC Type 3 was not only the predominant type (72%) present, but it also had a wide distribution, having been isolated at eight of the nine farms. Two other O-AGC types (O-AGC Type 2–1 and O-AGC Type 3–2) were identified, providing evidence of genetic variation. However, there was only partial concordance between the two serotyping techniques, which is likely linked to differences in the way serotypes are defined in the two approaches that were used. Nevertheless, in broad terms there is good evidence of intraspecific antigenic variation within our library of isolates, and collectively these data will be of crucial importance for assessing the pathogenicity of the isolates and the subsequent development of a vaccine for this emerging disease in New Zealand marine salmon farms.

Full text

Aquaculture 578 (2024) 740055
Available online 9 September 2023
0044-8486/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Serological and molecular typing of Tenacibaculum maritimum from New
Zealand farmed salmon, Oncorhynchus tshawytscha
Karthiga Kumanan
a
,
b
, Lizenn Delisle
a
, Connie Angelucci
c
, Ryan B.J. Hunter
a
,
Oleksandra Rudenko
e
, Jeremy Carson
d
, Richard N. Morrison
c
, Andrew C. Barnes
e
,
Kate S. Hutson
a
,
b
,
*
a
Cawthron Institute, Nelson, New Zealand
b
Centre for Sustainable Tropical Fisheries and Aquaculture, College of Science and Engineering, James Cook University, Townsville, Queensland, Australia
c
Centre for Aquatic Animal Health and Vaccines, Department of Natural Resources and Environment Tasmania (NRE Tas), Tasmania, Australia
d
Carson BioConsulting, Launceston, Tasmania, Australia
e
School of Biological Sciences and Centre for Marine Science, The University of Queensland, Queensland, Australia
ARTICLE INFO
Keywords:
Serotyping
Skin disease
Vaccine
Molecular typing
Tenacibaculosis
New Zealand
O-antigen
Chinook salmon
ABSTRACT
Tenacibaculum maritimum is a cosmopolitan bacterial pathogen with the potential to cause signicant losses in a
broad range of farmed and wild marine sh species. This study investigated the antigenic diversity of
T. maritimum isolated in culture from farmed Chinook (king) salmon (Oncorhynchus tshawytscha) from the
Marlborough Sounds in New Zealand. A total of 36 isolates were examined using antibody serotyping and rapid
molecular serotyping via multiplex PCR (mPCR) targeting genes encoding O-antigen biosynthesis enzymes.
Serological analysis using three different polyclonal antisera developed against T. maritimum isolated from
farmed Atlantic salmon (Salmo salar) in Tasmania, Australia revealed that there are three putative serotypes of
T. maritimum that occur in New Zealand. The predominant serotype was dened by a positive reaction to all three
Tasmanian antisera (antisera A, B and C), designated as serotype ABC. This serotype was found at all nine farm
locations tested and represented 81% of all isolates examined. The same library of isolates was evaluated by
mPCR serotyping and found three O-AGC types among tested isolates. O-AGC Type 3 was not only the pre-
dominant type (72%) present, but it also had a wide distribution, having been isolated at eight of the nine farms.
Two other O-AGC types (O-AGC Type 2–1 and O-AGC Type 3–2) were identied, providing evidence of genetic
variation. However, there was only partial concordance between the two serotyping techniques, which is likely
linked to differences in the way serotypes are dened in the two approaches that were used. Nevertheless, in
broad terms there is good evidence of intraspecic antigenic variation within our library of isolates, and
collectively these data will be of crucial importance for assessing the pathogenicity of the isolates and the
subsequent development of a vaccine for this emerging disease in New Zealand marine salmon farms.
1. Introduction
Genus Tenacibaculum from the family Flavobacteriaceae contains
Gram-negative, lamentous-rod bacteria, which are present predomi-
nantly in the marine environment (Suzuki et al., 2001) and include
species pathogenic to many marine sh species (Avenda˜
no-Herrera
et al., 2006; Fern´
andez-´
Alvarez and Santos, 2018). Of the bacterial
pathogens belonging to the Tenacibaculum genus, Tenacibaculum mar-
itimum (formerly known as Flexibacter maritimus) is frequently isolated
from diseased sh globally and has been the subject of considerable
research since the late 1970s (Bridel et al., 2020; Hikida et al., 1979;
Lagadec et al., 2021), with several complete and draft genome sequences
now available (P´
erez-Pascual et al., 2017; Lopez et al., 2022a). T.mar-
itimum is one of the eight pathogenic species in the genus Tenacibaculum
responsible for causing tenacibaculosis and is of considerable economic
signicance in marine aquaculture (Avenda˜
no-Herrera et al., 2006;
Fern´
andez-´
Alvarez and Santos, 2018; Nowlan et al., 2020). Tenaciba-
culosis has been identied in >30 marine sh species, which is indica-
tive of the lack of strict sh-host specicity of T. maritimum (Table 1).
In New Zealand, tenacibaculosis is a priority emerging disease in
* Corresponding author at: Cawthron Institute, Nelson, New Zealand.
E-mail address: kate.hutson@cawthron.org.nz (K.S. Hutson).
Contents lists available at ScienceDirect
Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture
https://doi.org/10.1016/j.aquaculture.2023.740055
Received 1 May 2023; Received in revised form 28 July 2023; Accepted 3 September 2023

Aquaculture 578 (2024) 740055
2
Table 1
Susceptible marine shes to Tenacibaculum maritimum and known antigenic groups in each country.
Susceptible marine sh
species
Country Source and key references Known serotypes Known O-AGC type
Atlantic salmon Salmo salar Australia Farmed (Handlinger et al., 1997) O3 (Lopez et al., 2022a)
A, B, C (A. Angelucci, pers. comm.)
2–1, 3–0, 3–1, 3–2 (
Lopez et al., 2022a)
Canada Farmed (Ostland et al., 1999; Frisch et al., 2018; Bateman
et al., 2022)
ND ND
Chile Farmed (Apablaza et al., 2017) ND ND
Spain Farmed (Pazos et al., 1993; Avenda˜
no-Herrera et al., 2004) O4 (Lopez et al., 2022a) 4–0 (Lopez et al.,
2022a)
Scotland Farmed (Ferguson et al., 2009) ND ND
Ireland Farmed (Downes et al., 2018) ND ND
Chinook salmon
Oncorhynchus tshawytscha
New Zealand Farmed (Kumanan et al., 2022; Brosnahan et al., 2019;
Johnston et al., 2020)
AB, B, ABC (This study) 2–1, 3–0, 3–2 (This
study)
USA Farmed & wild (Chen et al., 1995) ND ND
Canada Wild (Bass et al., 2022) ND ND
Coho salmon Oncorhynchus
kisutch
Canada Wild (Bass et al., 2022) ND ND
Rainbow trout Oncorhynchus
mykiss
Australia Farmed (Handlinger et al., 1997) ND 2–1, 3–0, 4–0 (Lopez
et al., 2022a)
Chile Farmed (Valdes et al., 2021) ND ND
Sockeye salmon
Oncorhynchus nerka
Canada Wild (Bateman et al., 2022; D.F⋅O, 2020; Pacic Salmon
Foundation, 2022)
ND ND
Barramundi Lates calcarifer Singapore Farmed (Radah et al., 2015) ND ND
Vietnam Farmed (Dong et al., 2017) ND ND
Black bream Acanthopagrus
butcheri
Australia Wild (Handlinger et al., 1997) ND ND
Black sea bream
Acanthopagrus schlegeli
Japan Farmed (Masumura and Wakabayashi, 1977; Rahman
et al., 2015)
ND 1–0 (Lopez et al.,
2022a)
Black damselsh
Neoglyphieodon melas
Egypt Captive (Haridy et al., 2015) ND ND
Dover Sole Solea solea Scotland Farmed (McVicar and White, 1982) O2 (Lopez et al., 2022a) 2–1 (Lopez et al.,
2022a)
Greenback ounder
Rhombosolea tapirina
Australia Wild (Handlinger et al., 1997) ND ND
Gurnard Chelidonichthys
lucernus
Italy Farmed (Magi et al., 2007) O3 (Magi et al., 2007) ND
Gilthead seabream Sparus
aurata
Greece Farmed (Kolygas et al., 2012) ND ND
Spain Farmed (Avenda˜
no-Herrera et al., 2004) O1 (Avendano-Herrera et al., 2005b) 1–0, 1–1 (Lopez et al.,
2022a)
Japanese ounder
Paralichthys olivaceus
Japan Farmed (Rahman et al., 2015; Baxa et al., 1986) ND 3–0 (Lopez et al.,
2022a)
Korea Farmed (Jang et al., 2009) ND 2–1 (Lopez et al.,
2022a)
Lumpsucker Cyclopterus
lumpus
Norway Farmed (Småge et al., 2016) ND ND
Northern anchovy Engraulis
mordax
USA Wild (Chen et al., 1995) ND ND
Orbicular batsh Platax
orbicularis
French
Polynesia
Farmed (Lopez et al., 2022b; Alix et al., 2020) O1, O3 (Lopez et al., 2022a) 1–0, 3–0, 3–1 (Lopez
et al., 2022a)
Pacic sardine Sardinops
sagax
USA Wild (Chen et al., 1995) ND ND
Picasso triggersh
Rhinecanthus assasi
Egypt Captive (Haridy et al., 2015) ND ND
Puffer sh Takifugu rubripes Japan Not stated (Rahman et al., 2014) NUF1081 (Rahman et al., 2014) ND
Red sea bream Pagrus major Japan Farmed (Masumura and Wakabayashi, 1977; Rahman
et al., 2015)
O1, O2, O1/O2 (Lopez et al., 2022a) 1–0 (Lopez et al.,
2022a)
Rock bream Oplegnathus
fasciatus
Japan Farmed (Wakabayashi et al., 1986) ND ND
Sand tiger shark Carcharias
taurus
Italy Captive-bred (Florio et al., 2016) ND ND
Seabass Dicentrarchus labrax Greece Farmed (Kolygas et al., 2012) ND ND
France Farmed (Bernardet et al., 1994) O1, O3, O2 (Lopez et al., 2022a;
Avendano-Herrera et al., 2005a)
1–0, 3–1, 2–1 (Lopez
et al., 2022a)
Malta Farmed (Yardımcı and Timur, 2015) O1 (Lopez et al., 2022a) 1–1, 2–1, 3–0 (Lopez
et al., 2022a)
Turkey Farmed (Yardımcı and Timur, 2015) O1 (Yardımcı and Timur, 2015) ND
Sole Solea senegalensis Spain Farmed (Cepeda and Santos, 2002; Pi ˜
neiro-Vidal et al.,
2007; Vilar et al., 2012)
O1, O3 (Avendano-Herrera et al.,
2005b)
ND
Portugal Farmed (Mabrok et al., 2016) O3 (Mabrok et al., 2016) ND
Striped trumpeter Latris
lineata
Australia Wild (Handlinger et al., 1997) ND ND
Turbot Scophthalmus maximus Spain Farmed (Alsina and Blanch, 1993; Devesa et al., 1989;
Pazos et al., 1993; Pi˜
neiro-Vidal et al., 2007)
O2 (Avendano-Herrera et al., 2005b) 2–1, 3–0, 4–0 (Lopez
et al., 2022a)
Italy Wild (Magi et al., 2007) O3 (Magi et al., 2007) ND
(continued on next page)
K. Kumanan et al.

Aquaculture 578 (2024) 740055
3
farmed marine Chinook (king) salmon (Oncorhnychus tshawytscha) (see
Kumanan et al., 2022). Globally, New Zealand is the largest aquaculture
producer of Chinook salmon for a niche market, contributing >70% of
worldwide Chinook salmon production (New Zealand Salmon Farmers
Association, 2022; Araujo et al., 2021). Over 85% of the production of
this anadromous species is in sea-pens located in the South Island of New
Zealand. Since 2012, salmon mortalities in sea-pens located in the
Marlborough Sounds have been associated with gross pathology of
tenacibaculosis (Brosnahan et al., 2019; Norman et al., 2013; Ministry
for Primary Industry, 2020; Kumanan et al., 2022). The disease is
prevalent during the summer months when the seawater temperature
exceeds 16 ◦C. Affected Chinook salmon exhibit varying degrees of
epidermal damage, including ulcerative epidermal lesions, skin spots,
tail n erosion and gill erosion (Johnston et al., 2020). This clinical
pathology in New Zealand Chinook salmon is consistent with typical
tenacibaculosis, which is characterized by a wide range of lesion
severity from small areas of scale loss to ulcerative skin conditions
exposing skeletal muscles (Frisch et al., 2018). Targeted surveillance
using optimized diagnostic techniques has revealed that T. maritimum
and other Tenacibaculum spp. are readily isolated from lesions of mori-
bund sh (Kumanan et al., 2022). Signicantly, there is an increasing
number of reports of marine sh species susceptible to tenacibaculosis
and a paucity of serological information (Table 1). These factors
alongside the emergence of tenacibaculosis in New Zeland farmed Chi-
nook salmon (Brosnahan et al., 2019; Kumanan et al., 2022) indicate a
growing need to identify T. maritimum serotypes to guide appropriate
disease prevention in New Zealand.
In many countries, the use of antimicrobials has been the primary
treatment option to manage tenacibaculosis (Nowlan et al., 2021; Irgang
et al., 2021). However, in line with the New Zealand Veterinary Asso-
ciation’s goal for primary production to be antibiotic free by 2030 (New
Zealand Veterinary Association, 2019), the New Zealand salmon in-
dustry do not use antibiotics (New Zealand King Salmon, 2016, High
Country Salmon, 2022, Sanford, 2022). There is concern that extensive
use of antimicrobials in aquaculture may increase the prevalence of
antimicrobial resistance (AMR) and thus reduce future treatment op-
tions and increasing the probability of AMR transfer to human patho-
gens (Zhao et al., 2021; Barnes et al., 2022; Preena et al., 2020; Reverter
et al., 2020; Bondad-Reantaso et al., 2023). Alternatively, vaccination is
an efcient disease prevention method that can be used to tackle AMR in
aquaculture (Bondad-Reantaso et al., 2023). At present, there are no
registered sh vaccines available in New Zealand. Internationally, there
is a single commercially available vaccine for prevention of
T. maritimum: ICTHIOVAC®-TM (serotype O2) vaccine for turbot,
Scophthalmus maximus, which is reported to provide short-term immu-
nity for six months (ICTHIOVAC® TM-Hipra Laboratories; Miccoli et al.,
2019).
Successful vaccination relies on the diligent selection of a strain(s) or
its antigen(s) (Barnes et al., 2022). A signicant challenge in developing
an effective vaccine against T. maritimum is the antigenic variability
present on the lipopolysaccharide (LPS) constituent of the outer mem-
brane of the pathogen (Van Gelderen et al., 2010; Avenda˜
no-Herrera
et al., 2004; Yardimci and Timur, 2016; Fern´
andez-´
Alvarez and Santos,
2018). LPS is an essential outer membrane constituent of Gram-negative
bacteria (Whiteld et al., 2020). The highly variable repeating
oligosaccharide units present in the immunogenic O-polysaccharide (O-
antigen) region of LPS results in the antigenically diverse O-specic
serotypes (Whiteld and Trent, 2014). Antigenic studies have revealed
the presence of at least four O-serotypes (O1, O2, O3 and O4) within the
species (Lopez et al., 2022a). Hence, a vaccine formulated against a
single strain of T. maritimum may not generate immunity against strains
with different O-antigen composition (Romalde et al., 2005). An effec-
tive vaccine should provide protection against a broad range of
T. maritimum strains that are present within major production regions
(Tinsley et al., 2011; Romalde et al., 2005; Barnes et al., 2022). This
would require a formulation of a multi-valent vaccine or a vaccine tar-
geting selected serotypes (incorporating the most virulent strains) to
efciently control the disease and prevent outbreaks (Barnes et al.,
2022). Multi-valent vaccines have successfully provided broad spectrum
protection against other antigenically heterogenous sh pathogens such
as Yersinia ruckeri (see Tinsley et al., 2011), Aeromonas hydrophila (see
Shirajum Monir et al., 2020) and Flavobacterium psychrophilum (see
Hoare et al., 2017). Therefore, investigating the diversity of
T. maritimum strains in discrete farming regions is needed to ensure that
effective autogenous vaccines are developed and deployed.
Using two bacterial typing methods, we aimed to identify the anti-
genic diversity of New Zealand T. maritimum isolates and compare them
to strains from Australia and other countries. First, conventional sero-
typing (antigen-antibody assay) was used to study serological relation-
ships between New Zealand isolates and serotypes of known
pathogenicity in the Tasmanian (Australia) Atlantic salmon industry
(Van Gelderen et al., 2010; C. Angelucci, NRE Tasmania, pers. com-
mun.). Second, we used the multiplex PCR (mPCR) based serotyping
scheme to detect the variation among genes of the O-antigen biosyn-
thesis cluster (O-AGC) of T. maritimum associated with salmon disease in
New Zealand (Lopez et al., 2022a). The variation present in the O-AGC
region is considered responsible for the diversity of the bacterial O-an-
tigen and the resulting multiple subtypes (Liu et al., 2021), and this
knowledge has enabled the comparison of local (New Zealand) O-AGC
variants with isolates from other countries and hosts for the rst time.
This type of assessment provides the basis on which emerging serotypes
in New Zealand marine farming environments can be characterized in
the future.
2. Materials and methods
2.1. Source and identication of bacterial strains
Tenacibaculum maritimum isolates were collected from nine Chinook
salmon farms in the Marlborough Sounds located on the north coast of
the South Island, New Zealand (Fig. 1). Four of the farms are in the
Pelorus Sound (sites A-D, Fig. 1), while ve farms are in Queen Charlotte
Sound (sites E- I, Fig. 1). The 36 isolates used in this study were collected
between January 2020 and August 2021 (Table 2). A total of 35
T. maritimum isolates were recovered in culture using Marine Shieh’s
Selective Medium (MSSM; Kumanan et al., 2022; Wilson et al., 2019)
from the skin of Chinook salmon; one isolate was recovered from
seawater. Isolates were assessed for colony morphology, which was
consistently pale white, irregular, at and strongly adhered to the sur-
face of the medium (Kumanan et al., 2022). To conrm the identity of
Table 1 (continued )
Susceptible marine sh
species
Country Source and key references Known serotypes Known O-AGC type
White seabass Atractoscion
nobilis
USA Farmed and wild (Chen et al., 1995; Drawbridge et al.,
2021)
ND ND
Yellowtail Seriola
quinqueradiata
Japan Farmed (Rahman et al., 2015; Baxa et al., 1986) ND 1–0 (Lopez et al.,
2022a)
Yellow eye mullet Aldrichetta
forsteri
Australia Wild (Handlinger et al., 1997) ND ND
Please consult key references for the various methodologies used to serotype isolates. ND: not determined.
K. Kumanan et al.

Aquaculture 578 (2024) 740055
4
bacterial isolates exhibiting a characteristic T. maritimum morphology
(Kumanan et al., 2022), genomic DNA (gDNA) was extracted using
AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany). Species-specic
primers targeting a 155 base pair (bp) region of the T. maritimum 16S
rRNA (forward 5
′
-TGCCTTCTACAGAGGGATAGCC-3
′
; reverse 5
′
-
CTATCGTTGCCATGGTAAGCCG-3
′
) were used in combination with the
detection probe (5’ HEX-CACTTTGG AATGGCATCG–BHQ1 3
′
; (Frin-
guelli et al., 2012) for PCR analysis as previously described (Kumanan
et al., 2022).
Three serologically distinct strains of T. maritimum obtained from the
Department of Natural Resources and Environment Tasmania (NRE
Tas), Australia and reference strains obtained from the National
Collection of Industrial, Food and Marine Bacteria (NCIMB) were
included in this study for comparative purposes (Table 2). The groups of
T. maritimum, isolated from Tasmanian farmed Atlantic salmon, were
dened based on a preliminary serotyping process, which recognized
two serotypes, designated A (TCFB 4635) and B (TCFB 3289) (L.
Schmidtke and J. Carson, unpublished data, 1995) and were subse-
quently expanded to include a third serotype designated C (TCFB 4574)
(see Table 3; C. Angelucci, NRE Tas).
2.2. Serotyping (dot-blot analysis)
2.2.1. Bacterial preparation
Tenacibaculum maritimum broth cultures were grown in 40 mL Ma-
rine Shieh’s Medium broth (Wilson et al., 2019) on a shaker at 180 rpm
for 48 h at 22 ◦C. In the event of bacterial aggregates (Fig. 2B), a new
broth preparation was made from a single colony and re-cultured until a
homogenous suspension was obtained. Microscopy of Gram-stained
samples from 48 h cultures was performed to check purity. Broth cul-
tures were transferred to sterile tubes and centrifuged for 30 min at 2900
xg at room temperature (RT). The supernatant was removed and the
bacterial pellet was washed twice using phosphate buffered saline (PBS)
(0.01 mol L
−1
, pH 7.4) by centrifugation at 2900 xg at RT. Washed
bacterial pellets were resuspended in PBS and formalin inactivated. Cell
integrity was assessed on Gram-stained cells under oil immersion at
1000×magnication using an Olympus BX5 microscope.
2.2.2. Antisera and dot-blot technique optimization
Rabbit antisera were developed to three Tenacibaculum maritimum
isolates from Tasmania, Australia (TCFB 3289, TCFB 4635 and TCFB
4574) using the services of the Walter and Eliza Hall Institute Antibody
Facility, Victoria, Australia. These isolates were prepared by culturing in
Marine Sheih’s Medium broth at 22 ◦C with shaking at 180 rpm for 24 h,
then cells were centrifuged (3000 rpm, RT, 15 min) and washed twice
with sterile PBS. Cells were formalin inactivated with 2% v/v formalin
and then washed again twice with sterile PBS (Angelucci, NRE Tas).
Each of these isolates represent a serotype and antisera were originally
assigned by NRE Tasmania as serotype A (anti 4635), serotype B (anti
3289) and serotype C (anti 4574) (see Table 3). A preliminary dot-blot
analysis conducted with minor modications made to the method
described by Avenda˜
no-Herrera et al. (2004) showed cross-reaction
Fig. 1. Tenacibaculum maritimum isolates were collected from moribund Chinook salmon (Oncorhynchus tshawytscha) in the Marlborough Sounds, New Zealand.
Isolates were obtained from nine farms: (A) Waihinau Bay, (B) Waitata, (C) Kopaua, (D) Forsyth, (E) Ruakaka, (F) Te Pangu, (G) Clay Point, (H) Ng¯
amahau,
(I) ¯
Ot¯
anerau.
K. Kumanan et al.

Aquaculture 578 (2024) 740055
5
Table 2
List of Tenacibaculum maritimum isolates used in this study.
O-AGC genetic
typing
Conventional serotyping using whole cell (formalin
inactivated)
Tenacibaculum maritimum
strains
Location Host Year
Dot-blot analysis using antisera to: Serotype
TCFB 4635
(A)
TCFB 3289
(B)
TCFB 4574
(C)
1 ++ − − A NCIMB 2153 Japan Acanthopagrus schlegeli 1976
++ ++ ++ ABC NCIMB 2154
T
Japan Pagrus major 1977
2–1 − + − B NCIMB 2158 Scotland Solea solea 1981
+ ++ − AB CCCM 125 Waitata, New Zealand * Oncorhynchus
tshawytscha
2021
+ + − AB CCCM 133 ¯
Ot¯
anerau, New Zealand
Y
Oncorhynchus
tshawytscha
2021
+ ++ ++ ABC CCCM 126 Waitata, New Zealand * Oncorhynchus
tshawytscha
2021
+ ++ ++ ABC CCCM 129 Clay Point, New
Zealand
Y
Oncorhynchus
tshawytscha
2021
+ ++ ++ ABC CCCM 139 Clay Point, New
Zealand
Y
Oncorhynchus
tshawytscha
2021
+ ++ ++ ABC CCCM 140 Ng¯
amahau, New
Zealand
Y
Oncorhynchus
tshawytscha
2021
+ ++ ++ ABC CCCM 152 Te Pangu, New Zealand
Y
Oncorhynchus
tshawytscha
2021
− ++ − B TCFB 3289~ Tasmania, Australia Salmo salar 2014
3–0 − ++ − B CCCM 013 Forsyth, New Zealand * Oncorhynchus
tshawytscha
2020
− ++ − B CCCM 015 Forsyth, New Zealand * Oncorhynchus
tshawytscha
2020
− + − B CCCM 103 Te Pangu, New Zealand
Y
Oncorhynchus
tshawytscha
2021
− + − B CCCM 127 Ruakaka, New Zealand
Y
Oncorhynchus
tshawytscha
2021
+ + − AB CCCM 105 Ruakaka, New Zealand
Y
Oncorhynchus
tshawytscha
2021
+ ++ ++ ABC CCCM 001 Waihinau, New Zealand
*
Oncorhynchus
tshawytscha
2020
+ ++ + ABC CCCM 004 Waihinau, New Zealand
*
Oncorhynchus
tshawytscha
2020
+ ++ + ABC CCCM 005 Waihinau, New Zealand
*
Oncorhynchus
tshawytscha
2020
+ ++ + ABC CCCM 006 Waihinau, New Zealand
*
Oncorhynchus
tshawytscha
2020
+ ++ ++ ABC CCCM 010 Waihinau, New Zealand
*
Oncorhynchus
tshawytscha
2020
+ ++ ++ ABC CCCM 014 Forsyth, New Zealand * Oncorhynchus
tshawytscha
2020
+ ++ + ABC CCCM 017 Forsyth, New Zealand * Oncorhynchus
tshawytscha
2020
+ ++ + ABC CCCM 018 Forsyth, New Zealand * Oncorhynchus
tshawytscha
2020
+ ++ ++ ABC CCCM 019 Forsyth, New Zealand * Oncorhynchus
tshawytscha
2020
+ ++ ++ ABC CCCM 020 Forsyth, New Zealand * Oncorhynchus
tshawytscha
2020
+ ++ ++ ABC CCCM 034 Forsyth, New Zealand * Oncorhynchus
tshawytscha
2020
+ ++ + ABC CCCM 035 Forsyth, New Zealand * Oncorhynchus
tshawytscha
2020
++ ++ ++ ABC CCCM 038 Forsyth, New Zealand * Oncorhynchus
tshawytscha
2020
+ ++ ++ ABC CCCM 039 Forsyth, New Zealand * Oncorhynchus
tshawytscha
2020
+ ++ ++ ABC CCCM 085 Waihinau, New Zealand
*
Oncorhynchus
tshawytscha
2020
++ ++ ++ ABC CCCM 101 Clay Point, New
Zealand
Y
Oncorhynchus
tshawytscha
2020
+ ++ ++ ABC CCCM 104 Te Pangu, New Zealand
Y
Oncorhynchus
tshawytscha
2021
+ + + ABC CCCM 106 Kopaua, New Zealand * Oncorhynchus
tshawytscha
2021
+ ++ + ABC CCCM 108 Ng¯
amahau, New
Zealand
Y
Oncorhynchus
tshawytscha
2021
++ ++ ++ ABC CCCM 122 ¯
Ot¯
anerau, New Zealand
Y
Oncorhynchus
tshawytscha
2021
(continued on next page)
K. Kumanan et al.

Aquaculture 578 (2024) 740055
6
across the collection of NZ strains with both antisera Anti-3289 and
Anti-4574. Subsequently, the antisera were cross-adsorbed and titrated
to increase the specicity of the antisera to their corresponding antigens
(see Supplementary text 1.1 and Supplementary g. 1.1). Second, since
there is a tendency for T. maritimum to auto-agglutinate (Fern´
andez-
´
Alvarez and Santos, 2018), we compared two methods of cell inactiva-
tion; formalin inactivation, using 0.5% formalin at RT and heat xed
(100 ◦C for 60 min, (Avenda˜
no-Herrera et al., 2004) using ve repre-
sentative isolates (see Supplementary text 1.2). The comparison estab-
lished that the method of inactivation did not inuence the dot-blot
outcome (see Supplementary text 1.2 and Supplementary Fig. 1.2).
Tenacibaculum maritimum isolates (Table 2) were enumerated using a
counting chamber (0.02 mm Helber Bacteria Counting Chamber,
Hawksley, UK) under 400×magnication, standardized to a cell count
of 1 ×10
9
cells/mL and held at 4 ◦C until used for serotyping. The cell
aggregates were dispersed using a Dounce homogenizer and cell diluted
1:40 in sterile PBS. The serological assays were carried out using the
method described by Avenda˜
no-Herrera with minor modications
(Avenda˜
no-Herrera et al., 2004) (Supplementary text 1.3) using whole
cell preparation inactivated with formalin. Dot blot reactions of un-
knowns were scored once a positive reaction was observed in the posi-
tive control (TCFB 4635, TCFB 3289 and/or TCFB 4574) of respective
antisera (Table 3). Reactions were scored according to colour intensity:
intense staining more intense or equal to the positive control were
recorded as “++”, medium staining as “+” and absence of reaction as
“-”. Each isolate was tested four times with each antiserum and corre-
sponding positive controls were included on each membrane (Table 3).
PBS and formalin inactivated Vibrio parahaemolyticus (NCTC 10884;
National Collection of Type Cultures) cells grown in marine broth at
22 ◦C for 48 h were used as a negative control.
2.3. O-AGC typing
Bacterial gDNA was extracted from 48 h cultures of T. maritimum
strains in Table 2 (excluding isolates originated from Tasmania),
following the method described in Section 2.1. The genetic loci within
the O-antigen genomic cluster (O-AGC) were amplied using primers
developed by Lopez et al., 2022a. O-AGC types of TCFB isolates were
predicted in silico from genome sequences deposited at PubMLST
database https://pubmlst.org/bigsdb?db=pubmlst_tenacibaculum
_isolates&page=query (id 133 =TCFB 3289; id 135 =TCFB 4574 and
id 137 =TCFB 4635). mPCR was performed for the rest of the isolates
mentioned in Table 2. Each mPCR reaction contained 10
μ
L of 1×MyFi
mix (BioLine, London, England), 0.5
μ
L of each primer (10
μ
M), 1
μ
L
gDNA (90–100 ng) and 5
μ
L of PCR grade water, and the cycling reaction
performed as described by Lopez et al. (2022a). To verify the test
method, positive controls NCIMB 2154
T
and NCIMB 2158 (Lopez et al.
(2022a) were included. The gDNA of other Tenacibaculum species iso-
lated from New Zealand Chinook salmon (i.e., T. dicentrarchi, CCCM20/
030 and T. soleae, CCCM20/023) were included to conrm the speci-
city of the primers towards T. maritimum. Five microliters of each PCR
product were electrophoresed (70 V for 45 min) on a 2% agarose gel
stained with 5% v/v RedSafe™-20,000×(iNtRonbio) and run in 1 X
Tris-acetate-EDTA (TAE) buffer along with 5
μ
L of AccuRuler 100 bp
DNA Ladder (MaestroGen). The bands of amplied genes were then
visualized using FireReader V10 (UVITEC, Cambridge, UK). To verify
the amplicons, mPCR products were puried using DNA Clean &
Concentrator-5 (ZymoResearch) and sequenced using Sanger technol-
ogy by an external contractor (Otago Genomics Facility, University of
Otago, Dunedin, New Zealand) using the PCR primers described in
Lopez et al. (2022a).
3. Results
3.1. Bacterial strain
All isolates had consistent colony morphology: pale white, at and
strongly adhered to the surface of MSSM. They were conrmed as
T. maritimum by species-specic PCR. Although most of the isolates
produced homogenous T. maritimum suspensions when cultured in
broth, some isolates formed bacterial aggregates (Fig. 2B), but this was
not specic to certain isolates and occurred sporadically. After 48 h of
incubation at 180 rpm, cultures yielded T. maritimum densities of
10
7
–10
9
cells/mL. Spherical cells (Fig. 2D) were seen occasionally in
Table 2 (continued )
O-AGC genetic
typing
Conventional serotyping using whole cell (formalin
inactivated)
Tenacibaculum maritimum
strains
Location Host Year
Dot-blot analysis using antisera to: Serotype
TCFB 4635
(A)
TCFB 3289
(B)
TCFB 4574
(C)
+ ++ + ABC CCCM 131 ¯
Ot¯
anerau, New Zealand
Y
Oncorhynchus
tshawytscha
2021
3–1 ++ − − A TCFB 4635~ Tasmania, Australia Salmo salar 2018
3–2 ++ ++ ++ ABC CCCM 102 Waihinau, New Zealand
*
Oncorhynchus
tshawytscha
2020
++ ++ ++ ABC CCCM 123 Ruakaka, New Zealand
Y
Oncorhynchus
tshawytscha
2021
+ ++ ++ ABC CCCM 128 Ruakaka, New Zealand
Y
Oncorhynchus
tshawytscha
2021
4 − − ++ C TCFB 4574~ Tasmania, Australia Salmo salar 2017
The notation * and
Y
indicate O. tshawytscha farms located at Pelorus Sound and Queen Charlotte Sound, respectively; superscript
T
is the type strain; TCFB =Tasmanian
Collection of Fish Bacteria; CCCM =Cawthron Culture Collection of Microorganisms (New Zealand isolates); NCIMB =National Collection of Industrial, Food and
Marine Bacteria; (−) faint detection; (+) medium detection; (++) strong detection.
Table 3
Tenacibaculum maritimum serotypes and respective antisera obtained from the
Department of Natural Resources and Environment Tasmania (NRE Tas),
Australia.
Reference materials Serotypes* Dilution and adsorbed / un-adsorbed
antisera used for dot-blot analysis
Isolate TCFB 4635 A NA
Isolate TCFB 3289 B NA
Isolate TCFB 4574 C NA
Rabbit anti- T. maritimum
(TCFB 4635) serum
Antiserum
A
1:64,000 (un-adsorbed)
Rabbit anti- T. maritimum
(TCFB 3289) serum
Antiserum
B
1:160,000 (adsorbed)
Rabbit anti- T. maritimum
(TCFB 4574) serum
Antiserum
C
1:20,000 (adsorbed)
*Serotypes ‘A’, ‘B’ and ‘C’ were as arbitrarily notated by the Centre for Aquatic
Animal Health & Vaccines, NRE Tas and were assigned independent of serotypes
or serotypes published elsewhere (unpublished data).
K. Kumanan et al.

Aquaculture 578 (2024) 740055
7
culture and have been observed by Pi˜
neiro-Vidal et al. (2012) in
T. dicentrarchi and Wang et al. (2008) in T. aiptasiae; spherical cells
appear to be a feature of lamentous bacteria described as ’exibacteria’
(Lewin and Lounsbery, 1969). Heat and formalin inactivation did not
affect the cell integrity of T. maritimum; however, fewer cell aggregates
were observed in heat inactivated samples compared to formalin inac-
tivated samples (Fig. 2C and D).
3.2. Dot-blot
Antisera developed for T. maritimum isolated from Atlantic salmon in
Tasmania bound T. maritimum isolates from Chinook salmon and the
reference cultures (NCIMB 2153, NCIMB 2154
T
and NCIMB 2158)
(Table 2). The specicity of each antiserum toward its homologous
positive control (Tasmanian Atlantic salmon isolates) and the reactions
with some representative New Zealand isolates of T. maritimum is given
is Fig. 3.
Serological analysis using the three antisera revealed some serolog-
ical diversity among New Zealand T. maritimum isolates (Table 2). Three
distinct serological groups were present among the isolates tested. Of the
36 New Zealand isolates examined in this study, 29 (81%) reacted with
all three antisera (antisera A, B and C: designated as serotype ABC),
three isolates (8%) were recognized by antisera A and B (designated as
serotype AB) and 4 (11%) were detected by antiserum B only (Fig. 4). All
three of these serological groups were detected in both Pelorus Sound
and Queen Charlotte Sound in the Marlborough Sounds region (Fig. 4).
Serotype ABC was present at all nine farms and was the predominant
type of isolate tested in Chinook salmon farms in the Marlborough
Sounds. This antigenic combination was also observed for the reference
isolate NCIMB 2154
T
isolated from Japan (Pagrus major), but not the
isolates from Scotland or Australia (Table 2). Dot-blot analysis revealed
the presence of serotype B in New Zealand, which is also present in
Australian Atlantic salmon (TCFB 3289) and Dover sole in Scotland
(NCIMB 2158) (Table 2).
3.3. Molecular typing/mPCR
The mPCR assay performed on 36 New Zealand T. maritimum isolates
identied three O-AGC types: Type 2–1, 3–0 and 3–2 groups in Chinook
salmon farms (Table 2 and Fig. 5). Of the 36 isolates tested, 26 isolates
(72%) were classied as O-AGC Type 3, which was predominant in the
Pelorus Sound and Queen Charlotte Sound sites (Fig. 4) and found in all
farms except for one. The second O-AGC group of Type 2–1 corre-
sponded to seven isolates (19%) across ve farms in both Pelorus and
Queen Charlotte Sounds (Fig. 4). The nal group, O-AGC Type 3–2,
corresponded to three isolates (8%). All three O-AGC groups were pre-
sent in Pelorus Sound and Queen Charlotte Sound (Fig. 4). mPCR typing
of NCIMB 2154 and NCIMB 2158 is consistent with typing by Lopez et al.
(2022a) and provides condence in the O-AGC typing results of the New
Zealand T. maritimum isolates (Fig. 5). The T. maritimum mPCR assay
was specic to T. maritimum, as no product from T. soleae (CCCM20/
023) or T. dicentrarchi (CCCM20/030) gDNA was observed in either the
singleplex or mPCR for the T. maritimum O-AGC construct (data not
shown). The amplicons were conrmed to be the correct target gene by
Fig. 2. (A and B) Examples of Tenacibaculum mar-
itimum culture grown in broth with the same incu-
bation conditions. (A) Broth exhibiting homogenous
suspension of T. maritimum. (B) Isolate that formed
bacterial aggregates. (C and D) Gram-stained cells of
T. maritimum under oil immersion at 1000×magni-
cation. (C) Heat inactivated cells showing minimal
aggregation. (D) Formalin inactivated cells showing
multicellular clumps. The arrow in gure D indicates
a spherical cell of T. maritimum.
K. Kumanan et al.

Aquaculture 578 (2024) 740055
8
Fig. 3. Dot-blot serotyping showing the specicity of each antiserum toward its positive control (TCFB 3289, 4635 and 4574). Representative New Zealand
Tenacibaculum maritimum isolates (CCCM 005, 006, 010 and 017) detected by all three antisera (A, B and C); isolate CCCM 013 was only detected by Antiserum B.
Fig. 4. The relative percentage of Tenacibaculum maritimum serological and O-AGC (mPCR) groups present in the library of isolates collected from Chinook salmon
(Oncorhynchus tshawytscha) farms in the Marlborough Sounds, New Zealand (total 36 isolates).
K. Kumanan et al.

Aquaculture 578 (2024) 740055
9
Sanger sequencing technology.
4. Discussion
The increasing incidence of T. maritimum associated mortalities in
New Zealand (Kumanan et al., 2022; Brosnahan et al., 2019) along with
New Zealand’s warming seas threaten the Chinook salmon industry
(NIWA, 2022; Lane et al., 2022). To mitigate this emerging infectious
disease threat (Kumanan et al., 2022; Brosnahan et al., 2019), a timely,
reliable and effective prevention strategy is required. As a rst step to-
ward disease prevention, we have taken a pragmatic approach to study
the antigenic properties of T. maritimum. This is the rst study reporting
the antigenic diversity of T. maritimum isolated from Chinook salmon
exhibiting signs of tenacibaculosis in New Zealand. Utilizing antisera
from Tasmania, we found at least three T. maritimum serotypes (ABC, AB
and B) among the isolates tested in this study. There are also multiple
T. maritimum serotypes that have been identied in isolates from
Atlantic salmon (C. Angelucci, NRE Tasmania, pers. comm.; Van Gel-
deren et al., 2010). Originally, T. maritimum serotypes were thought to
be host specic (Avenda˜
no-Herrera et al., 2004); however, increased
investigation of this pathogen among multiple vulnerable host species
has identied that serotypes are not strictly host specic (Lopez et al.,
2022a; Van Gelderen et al., 2010). Our results align with these previous
ndings, as our data show antigenic variation in isolates obtained from a
single species, Onchorhynchus tshawytscha.
A total of 29 T. maritimum isolates (81%) from New Zealand Chinook
salmon were positive with all three Tasmanian antisera (antisera A, B
and C), and were allocated to serotype ABC. This suggests that the
majority of New Zealand T. maritimum isolates share antigenic compo-
nents with each of the three Tasmanian T. maritimum serotypes. This
novel serotype occurred across all Chinook salmon farms located at
Pelorus Sound and Queen Charlotte Sound in New Zealand and was not
previously reported to occur in Tasmania (C. Angelucci, pers. comm.).
We also noted that isolate NCIMB 2154
T
, which was isolated from
diseased red seabream in Japan, was also assigned to serotype ABC. In
addition, we found New Zealand isolates that only typed as serotype B,
and these were detected in eight of the nine farms; the reference isolate
NCIMB 2158 from Dover sole, Scotland, also tested as serotype B. Iso-
lates specic to serotype B have also been reported in Tasmanian
Atlantic salmon (C. Angelucci, pers. comm.). Notably, the antigenic
variability reported in NCIMB 2154 in this study has previously been
observed in an O-antigen serotyping scheme where this isolate was
assigned to serotype O1 and O2 (Avenda˜
no-Herrera et al., 2004;
Fern´
andez-´
Alvarez and Santos, 2018). However, NCIMB 2158 was spe-
cic to serotype B in this study and was only assigned to group O2 in a
previous study by Avenda˜
no-Herrera et al. (2004).
Three isolates from New Zealand Chinook salmon were designated as
serotype AB, as were some isolates from Tasmanian Atlantic salmon (C.
Angelucci, pers. comm.); none of the NCIMB reference strains were
identied as serotype AB. Serotype C was based on an isolate from
Tasmanian Atlantic salmon, and none of the New Zealand or NCIMB
strains were assigned to this serotype. Based on the samples tested,
serotype C appears to be unique to Tasmanian Atlantic salmon.
Serotyping by antigen-antibody reaction of T. maritimum isolates
provides insight into phenotypic strain variation based on surface anti-
gens of New Zealand isolates from Chinook salmon. This forms the basis
for comparing Atlantic salmon isolates in Tasmania with isolates from
Chinook salmon in New Zealand. Nevertheless, antibody-based sero-
typing schemes can encounter drawbacks, including the selection of
suitable strains for antiserum production, establishing specicity along
with choice of assay method, subjective interpretation, and standardi-
zation and harmonization of serotyping schemes (Lopez et al., 2022a;
Sloan et al., 2017). For example, in this study, we found it difcult to
produce homogenous bacterial preparations for dot-blot assay because
of the tendency of T. maritimum to sporadically form cell aggregations, a
problem that was also observed by Rahman et al. (2014). Bacterial
occulation, cell clumping and strong adherence to culture asks added
further difculties when estimating cell densities. Serotyping of
T. maritimum isolates by antigen-antibody reaction in this study de-
scribes types with indistinct boundaries. Consequently, allocation of the
New Zealand strains of T. maritimum to particular serotypes should be
considered preliminary; however, our results do provide relevant
phenotypic information as to the likely strain variation and distribution
across different farms and production areas.
Recent sequencing of the T. maritimum whole genome (Bridel et al.,
2020; P´
erez-Pascual et al., 2017) has conrmed the use of genomic loci
encoding antigenic variability as a means of establishing less ambiguous
serotyping (Lopez et al., 2022a). The variation in the O-AGC genomic
cluster of T. maritimum among available whole genome sequences dis-
tinguishes eight O-AGC subtypes, which fall into four major O-AGC
Fig. 5. Representative genetic prole of Tenacibaculum maritimum O-AGC groups by mPCR present in New Zealand Chinook salmon (Oncorhynchus tshawytscha)
farms; data for Tenacibaculum maritimum isolates held in Cawthron’s Culture Collection of Microorganisms (CCCM). Reference T. maritimum cultures were obtained
from the National Collection of Industrial, Food and Marine Bacteria (NCIMB) and used as positive controls.
K. Kumanan et al.

Aquaculture 578 (2024) 740055
10
types corresponding to four serotypes (Lopez et al., 2022a). We assessed
the suitability of this rapid proling system on our collection of New
Zealand T. maritimum isolates. Molecular subtyping revealed three O-
AGC types (Type 2–1, Type 3–0 and Type 3–2) across the 36 New Zea-
land T. maritimum isolates that were analyzed. Of the three O-AGC
groups identied in New Zealand, Type 3–0 was the most abundant
(72%) across both Pelorus and Queen Charlotte Sounds. Tenacibaculum
maritimum of sequence Type 3–0 were also isolated from farmed Tas-
manian Atlantic salmon, orbicular batsh (French Polynesia), olive
ounder (Japan) and turbot (Malta and Spain) (Lopez et al., 2022a). The
globally predominasnt subtype 2–1 was the next most abundant type
comprising 20% of New Zealand isolates that were isolated from Pelorus
and Queen Charlotte Sounds. This subtype has been reported to occur in
a variety of locations and hosts including Dover sole from Scotland
(NCIMB 2158), white seabass (USA), turbot (France and Spain), Euro-
pean seabass (Malta, France, Italy and Spain), orbicular batsh (French
Polynesia), Atlantic salmon (Australia), rainbow trout (Australia) and
olive ounder (South Korea) (Lopez et al., 2022a). O-AGC subtype 3–2
was detected in only 8% of New Zealand isolates from Pelorus Sound
and Queen Charlotte Sound. Moreover, this subtype has only been
detected in New Zealand Chinook salmon, Atlantic salmon and striped
trumpeter from Australia (Lopez et al., 2022a). The wide range of sh
species from which these O-AGC types of T. maritimum have been iso-
lated indicates there is no host or geographical specicity associated
with the O-AGC types.
In this study, serotype ABC was found across all three O-AGC types
(Type 2–1, 3–0 and 3–2) identied in the 36 New Zealand isolates of
T. maritimum that were tested. Contrary to the expectations of the pro-
posed typing scheme by, Lopez et al. (2022a), we did not see a clear
correlation between antibody-based serotyping and O-AGC typing in
this study. For example, NCIMB 2153 and 2154
T
were O-AGC Type 1,
but different serotypes were assigned by antibody-based assay. This
indicates that factors other than LPS O-antigen may play a role in
serological classication (Fratamico et al., 2016). It has long been
known that multiple antigens such as somatic (O-antigen), agella (H-
antigen) and capsular polysaccharide [K-antigen/capsular poly-
saccharide (CPS)] comprise the bacterial envelope and serological
assignment of bacteria is inuenced by factors such as antigen expres-
sion, immunodominance and availability (Orskov et al., 1977; Rochat
et al., 2017; Stenutz et al., 2006; Whiteld and Roberts, 1999). Bacterial
LPS and CPS are both involved in producing an immunological response
in the host (Perera et al., 2021; Evrard et al., 2010; Zhang et al., 2019).
CPS has been widely studied in some bacterial species, including Kleb-
siella and Salmonella for antigenic typing (Choi et al., 2020; Perera et al.,
2021; Wyres et al., 2016). Notably, LaFrentz et al. (2007) reported two
distinctive carbohydrate-banding patterns correspond to the LPS and
CPS in F. psychrophilum, a closely related species to T. maritimum and
member of the family Flavobacteriaceae. It was also suggested the CPS is
an important virulence factor for the cell adherence of F. psychrophilum.
To the best of the authors’ knowledge, there are no detailed published
reports about the CPS region of Tenacibaculum maritimum although the
presence of a capsular structure was reported by Avenda˜
no-Herrera
(2005) using electron microscopy. In this study, antisera developed
against whole inactivated T. maritimum was used for serotyping, and one
would expect the antisera would recognize all antigenic components
present on the cell surface of bacteria, including LPS (O) and CPS (K),
which contrasts with the O-AGC mPCR typing that is limited to O-an-
tigen (Lopez et al., 2022a). We suspect, the absence of K:O based genetic
typing may have contributed to the lack of concordance between sero-
typing and O-AGC mPCR types observed in this study as well as in
F. psychrophilum isolates as reported previously (Rochat et al., 2017).
Given the nature of T. maritimum to produce exopolysaccharides, it is
essential to further investigate the potential involvement of CPS in
parallel with LPS as described in Klebsiella species (Wyres et al., 2016;
Kubler-Kielb et al., 2013). The known virulence mechanism of CPS to
evade host innate immunity by its antiphagocytic ability (Pettis and
Mukerji, 2020) should not be ignored in determining serotype and
pathogenicity, and CPS diversity should be accounted for during a
vaccine development (Lin et al., 2022; Barai et al., 2022). Nevertheless,
this preliminary study has value because the antigenic relationship of
T. maritimum between Australia and New Zealand has been assessed, and
importantly we have identied the predominant serotype in New Zea-
land Chinook salmon, a nding that will aid the epizootiological
assessment of disease outbreaks.
In conclusion, our study reports the rst antigenic characterization of
T. maritimum associated with Chinook salmon exhibiting clinical signs of
tenacibaculosis. Antigenic variation identied by both antibody and
mPCR based typing will allow us to investigate whether there is an as-
sociation between serotype assignment and the degree of T. maritimum
virulence through a pathogen challenge model, which is essential for
appropriate strain selection for an effective vaccine (Barnes et al., 2022;
Tinsley et al., 2011; Shirajum Monir et al., 2020; Hoare et al., 2017).
Results obtained from this work will enable the formulation of an
autogenous vaccine comprising local antigens. This approach is ex-
pected to be more effective and efcient than using an existing com-
mercial vaccine, which may not be serotype specic and can be subject
to extended delays due to regulatory and policy approval. Critically,
such imported vaccines may not be effective against local or emerging
serotypes (Barnes et al., 2022). It is important to note that antigenic
characterization of a sh pathogen is not a one-time analysis; instead, it
should be a continuous process as part of disease surveillance in aqua-
culture to enable the detection of emerging serotypes that may cause
disease in immunized sh (Barnes et al., 2022). The mPCR typing in
parallel with serological techniques can serve as a time-effective
screening tool to monitor emerging T. maritimum variants.
Declaration of Competing Interest
The authors have no competing interests to declare.
Data availability
Link will be provided a proof stage
Acknowledgments
This research was funded by the Royal Society Te Ap¯
arangi: Catalyst
Seeding Fund (CSG-CAW2001) and the New Zealand Ministry of Busi-
ness, Innovation and Employment (Aquaculture Health to Maximise
Productivity and Security; CAWX1707). Kumanan was supported by an
Australian Government Research Training Program (RTP) scholarship.
Seumas Walker organized and oversaw eld sampling. Zac Waddington
and Stuart Barnes assisted in sample collection. Mark Engleeld coor-
dinated sample reception and laboratory logistics. Carol Peychers and
Chaya Bandaranayake provided technical support. Lisa Floerl and Eden
Cartwright (Bird Circus) assisted with graphics (Figs. 1 and 2, respec-
tively). None of the authors have conicting industrial links or
afliations.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.aquaculture.2023.740055.
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