2.1. Disease outbreak, bacterial isolation, and histopathology
In
late May and June 2018,
following
continuous high temperatures and a lack of
rain (since early March), mass
mortalities of juvenile Pacific abalones were
reported
in many aquaculture farms
in Dongshan country,
Fujian province, P. R. China.
During
our ensuing investigation, we found extremely high mortality rates,
reaching 100% in some
of
the most affected farms. Affected
abalone tended to gather at the
tops of the
concrete
blocks in the morning,
and exhibited weak pedal attachments
to the substrate; the pedal epithelial cells were also bleached
(Fig. 1A). As many moribund and
healthy
individuals as possible were collected and transported to our laboratory
under refrigeration for further analysis.
In
the laboratory, diluted mucus was collected from the body surface.
Several tissues (including the pedal, mantle, gill, hypobranchial gland,
gut,
and digestive gland) were
homogenized after dissection and washing with 75% ethyl alcohol to
reduce the risk of contamination. The mucus dilutions and tissue
homogenates were cultured at 28 °C
for 48 h in 2216 E agar culture medium.
Individual
colonies were then purified
by
the streak plate method and kept frozen in 15% glycerol at
-80
°C.
The tissues listed above were fixed
for pathological examination. The primary aquatic environmental
parameters at each farm were measured at 7:00 A.M. using a
portable
multiparameter water quality
measuring instrument (AZ86031, AZ Instrument, China) at our test site,
the average water temperature was
28.3 °C,
pH
was 7.6, salinity was 35.3, and
dissolved
oxygen was 6.3 (Fig. 1B).
2.2
16S rDNA gene sequence
analysis
We
used the cooking method to release genomic DNA from
200
μL of fresh bacterial liquid. Briefly,
after centrifugation and washing
twice with sterile deionized water
(SDW),
bacterial colonies were resuspended in the 100 μL SDW and boiled at 98
°C for 10 min. The supernatants were then used for PCR amplification as
soon as possible.
PCR
products were
amplified
with universal primers (27 F and 1492 R; and purified using Promega
Wizard SV Gel and PCR Clean-up System.
DNA
sequences
were analyzed using NCBI Blastn
against
the EzBioCloud
database
after being sequencing by BORUI Bio-Technology Co., Ltd (Xiamen, China).
A
phylogenetic
tree was constructed using
MEGA
5.0 (Tamura et al., 2011) and iTOL v5 (Letunic & Bork, 2019).
Differences
in the distributions of culturable bacteria between moribund and healthy
individuals were determined based on taxonomic identifications.
2.3Bacterialresistancein
vitro
To investigate bacterial
resistance
in vitro, mucus was
collected
following previous studies, with some modifications (Adel et al., 2016;
Lv et al., 2019; Valero et al., 2019).
Briefly, mucus from the abalone
body surface (2.5 ± 0.2 cm in length)
was
obtained by quickly rinsing the
animal with 2 mL
filtered
seawater
(FSW).
We ultimately collected about 1 mL viscous mucus
from each individual pedal. The
collection process was completed rapidly. The collected body surface
mucus was immediately transferred to a sterile microcentrifuge tube and
kept on
ice.
Sterile mucus
was
filtered through
a
0.22 μm filter to remove bacteria. Protein concentration in each mucus
sample was measured using a Pierce BCA Protein Assay Kit, and then
adjusted to 0.01 μg / μL protein. Adjusted samples were
stored at -80
°C.
Bacterial
species were collected during the logarithmic growth phase, when the
optical
density (OD) value at 600 nm
reached 0.6–0.8, as determined by a microplate reader (Infinity Pro200
Pro spectrophotometer, Tecan, Spain). Collected bacteria were washed
twice with
FSW.
We
prepared 10 × 106 bacteria (Sanahuja et al., 2019)
based
on
flow
cytometry (Cytoflex, Beckman, USA),
and
transferred
the bacterial solutions to the mucus, to a
final
volume of 200
µL.
Three
replicates of different mixtures were added to a 96-well plate and
incubated at
28
°C.
FSW
were used as the control.
The
OD value of each mixture at 600 nm was recorded after 0, 30, 60, 90,
120, and 240 min. Bacterial strains with greater growth in mucus were
more resistant and more likely to be pathogenic; these strains were
selected as candidates for virulence study in vivo .
Bacterial growth was also
assessed
in 2216 E liquid medium at 28 °C in order to exclude
strains with higher growth rates at
this temperature. Strains with high resistance to mucus were also
selected to determine the optimum survival temperature (26 ºC,
28
ºC, or 30 °C). The
OD value was recorded every hour
for
12
h.
2.4Thepathogenicity
ofthe
candidate isolatesin vivo
To
confirm
the
pathogenicity of each candidates,
experimental challenges were performed using healthy abalone (seven
months old; average shell length
2.5 ± 0.2 cm) obtained from an
abalone farm in Jinjiang, Fujian Province,
China.
Prior to bacterial challenge, abalones were
randomly
allocated among tanks (20 animals per treatment) containing 10 L
FSW
and
allowed
to acclimatize to laboratory conditions for at least for a week before
experimentation.
After
the acclimation period, the water temperature was raised to 28 ºC, at a
rate of 0.5 ºC per day, using the constant-temperature system.
Candidates
were cultured and washed as described above. To test each candidate, 140
abalones were randomly divided among 7 groups (6 infected groups and 1
control group). Each infected group was immersed in 1 of 6 bacterial
solutions:
1.0
× 103 CFU mL-1, 1.0 ×
104 CFU mL-1, 1.0 ×
105 CFU mL-1,
1.0 × 106 CFU
mL-1, 1.0 × 107 CFU
mL-1, and 1.0 × 108CFU
mL-1.
The control group was untreated. The same volume of water in each tank
was replaced with FSW every 24 h
during the experiment. Clinical symptoms and
mortality rate in each group were
recorded daily for 10 days to calculate the 50% lethal dose (LD 50). In
order to validate Koch’s postulate, bacteria were re-isolated and
identified from the moribund abalones. The pathogenicity of each
candidate isolate was also tested at 25 °C.
2.5Antibioticsensitivity
testing
We
used the
disc
agar diffusion method to test the antibiotic
sensitivity
of each isolate. Resistance was assessed according to the guidelines of
by the British Society for Antimicrobial Chemotherapy (BSAC).
Antibiotic
sensitivity was recorded as either antimicrobial susceptibility (S),
medium susceptibility (I), or resistant (R), based on the diameters of
the inhibition zones on 2216 E agar
plates. Drug resistance was assayed
using 26 antibiotics (Hangzhou Microbial Reagent Co., Ltd., China):
4
penicillins,
6
cephalosporins,
2
quinolones,
4
aminoglycosides,
3
tetracyclines, 3
macrolides,
a
glycopeptide,
an
amphenicol,
a
nitrofuran, and
a
polypeptide.
The
multiple
antibiotic resistance index (MARI) of each isolate was calculated as a/b
*100, where a was the number of antibiotics to which the particular
isolate was resistant, and b was the number of antibiotics to which the
isolate was exposed (Zhu, Dong, Weng, & He, 2018). The resistance rate
of each isolate was also calculated.
3. RESULTS
3.1 Differences in the distributions ofculturable
bacteria
We
isolated and identified 93
bacterial
strains, of which 54 were isolated
from
moribund
abalone and 34 were isolated from
healthy
abalone.
The
16S rDNA sequences were taxonomically identified based on the NCBI
database. In both healthy and moribund abalone, most isolates
fell into the phylum
Proteobacteria (class
Alphaproteobacteria
and Gammaproteobacteria), followed by
Bacteroidetes
(14%).
We identified five
bacterial
families in the moribund abalone only (Moraxellaceae, Kiloniellaceae,
Ferrimonadaceae, Flammeovirgaceae, and Halomonadaceae), and three
families in the healthy family only (Alteromonadaceae,
Oceanospirillaceae, and Oleiphilaceae). Five
families
(Vibrionaceae, Shewanellaceae, Pseudoalteromonadaceae,
Flavobacteriaceae, and Rhodobacteraceae) were shared
between
the
healthy and
the
moribund
individuals. The abundance
of
the culturable Pseudoalteromonadaceae, with the exception of the
Vibrionaceae and the Shewanellaceae,
was
much greater in the
moribund
abalone
(28%) as compared to the healthy abalone (5%), while
the
abundances of the Flavobacteriaceae and Rhodobacteraceae
dropped
drastically (to 5%) in the moribund abalone.
The
culturable
Pseudoalteromonadaceae
included
15
isolates
in
the
moribund abalone and two isolates
in
the
healthy
abalone.
Comparison with the culturable 16S rDNA gene sequences in the EzBioCloud
database showed that 14 strains isolated from the moribund abalone
(SDCH01, SDCH02, SDCH03, SDCH27, SDCH28, SDCH29, SDCH31, SDCH87, SDCH90,
SDCH91, SDCH92, SDCH93, SDCH95, and
SDCH97) and one strain from the
healthy abalone (SDCH40) were 99.18–99.68% similar toP.
shioyasakiensis SE3 (T) (JCM 18891). Remarkably, the abundance of
strainP.
shioyasakiensis SE3 (T) was 26% in the moribund abalone as compared to
2.5%
in
the healthy abalone (Fig. 2A).
Retrospective
analysis showed that, of
the 14 P. shioyasakiensisSE3 (T) strains isolated from the
moribund abalone,
eight
strains
(SDCH01,
SDCH02,
SDCH03, SDCH27, SDCH28, SDCH29, SDCH31, and SDCH87)
were
isolated from mucus, four strains
(SDCH90, SDCH91, SDCH92, and
SDCH93)
were isolated from the foot, strain SDCH95 was isolated from the gut,
and
strain
SDCH40 was isolated from the digestive gland. In the healthy abalone,P. shioyasakiensis SE3 (T) strain SDCH40 was isolated from the
gill (Fig. 2B).
3.2
TheresistanceofPseudoalteromonadaceaein vitro
After
co-incubation with fresh mucus and FSW, mixture absorbance was
measured for 240 min. In the FSW
group, strains did not differ
significantly over time (Fig. 3A).
In the mucus group, the resistance
of the strains identified as P. shioyasakiensis SE3 (T) to
abalone mucus differed noticeably (Fig.
3B):
strains were either
mucus-resistant,
unaffected, or mucus-sensitive. The OD of
the mucus did not differ
significantly over time. Strains SDCH28, SDCH29, SDCH40, and SDCH93 were
considered mucus-sensitive, due to the significant decrease in OD within
the first 30 minute (p < 0.05). Strains SDCH1, SDCH3, SDCH27,
SDCH31, SDCH91, SDCH92, SDCH, and SDCH95 were considered unaffected, as
OD did not change during the first 120 min.
Strains
SDCH87, SDCH90, and SDCH2
were
considered mucus-resistant, because OD increased significantly within 60
min
for
SDCH87 and SDCH90
(p
< 0.05), and within 90 min for SDCH2 (p < 0.05).
Several strains had high growth
rates in 2216E liquid medium at 28°C but did not exhibit
mucus-resistance, including SDCH29,
SDCH92, and SDCH93,
indicating
that the high growth rates of
SDCH87,
SDCH90, and SDCH2 in mucus were a
result of mucus-resistance, not the temperature increase (Fig. 3C).
The
optimum temperature for both SDCH87
and SDCH90 was 28°C (Fig. 3D),
consistent with water temperatures during the disease outbreak. Thus,
SDCH87 and SDCH90 were selected as candidate pathogens for the bacterial
challenge.
3.3 SDCH87, a
candidate pathogen, induced disease in
vivo at 28°C
The responses of juvenile H. discus hannai to
bacterial
exposure were assessed to validate our in vitrobacterial
resistance
results.
We found that strain SDCH87
had
outstanding resistance to mucus as well as obvious
lethal effects on abalone.
Abalone survival rates decreased in
a dose-dependent manner: abalones
presented
the same clinical signs after
exposure to higher
bacterial
doses
(more than 1.0 ×
104 CFU mL-1). The LD50 value of
SDCH87 was 5.6 ×
105CFU
mL-1 at 28°C
(Fig.
4).
This bacterial strain was re-isolated from the mucus samples from
moribund and dead abalone. The results indicated that juvenile abalone
mortality was caused by SDCH87.
In addition, almost
all
abalone deaths occurred within two
days of the challenge, and no obvious tissue injuries were observed with
the naked eye. Therefore, these deaths were caused by acute bacterial
infection. However, no abalone mortality was associated with identical
exposure to SDCH90 or any other strain.
Importantly, strain SDCH87
didn’t show pathogenicity towards
Pacific abalone at 25°C, based on
the fact that no deaths were recorded at this temperature.
3.4Antibiotic
sensitivity ofthe
15 P. shioyasakiensis strains
More
than 60%
of
the 15 P. shioyasakiensis strains exhibited strong resistance to
penicillins, cephalosporins, aminoglycosides, polypeptides,
glycopeptides, tetracyclines, macrolides, and nitrofurans (Table 1). All
strains showed complete resistance to oxacillin, ceftriaxone, kanamycin,
neomycin, amikacin, and tetracycline. Only
chloramphenicol
was able to affect all strains (Table 1). The
MARIs
of all strains were high, ranging from 35% to 96%. The
MARIs
of
SDCH
3 and
SDCH27
were the lowest (35% and 54%, respectively). The highest
MARI
was recorded for strain SDCH31
(96%), while the MARIs of the remaining strains were 73–92%. These
results indicated that multiple antibiotic resistance was common across
the bacterial strains isolated from cultured abalone.
3.5Phylogenetic
analysis based on 16SrDNA
To investigate the relationships among the 15P.
shioyasakiensis strains isolated herein, as well as 60
previously-published sequences ofP. shioyasakiensis available from the NCBI (selected based on 16S
rDNA sequence similarity), we
constructed
a neighbor-joining phylogenetic tree. Strains
SDCH25
and SDCH61 isolated herein were used as outgroups
(Fig 5).
With the exception of
strain SDCH29, all of the other
strains isolated herein clustered
together. Interestingly,Pseudoalteromonassp. strain
M14-00202-5E
(KY229817.1), which was isolated from Pacific oysters during an
investigation of mass mortalities of unknown etiology (Go et al., 2017),
also fell into this cluster. The 16S
rDNA gene sequences of 17 strains isolated herein were submitted to the
GenBank database (accession numbers MT912612 to MT912618).
3.6Histopathology
Histopathological changes were observed in the pedal tissues of moribund
abalone. Pathological analyses showed
that, in the moribund abalone,
mucus secretion from the epithelial
cells decreased, secretory cells vacuolization occurred, epithelial cell
penetrability increased, and the connective tissue became loose,
destroying the integrity of the
pedal mucosal tissue (Fig 6). Histopathological observation identified
no abnormalities in any other
tissues. In addition, parasites, fungi, and virus were not detected on
the body surface. Therefore, we suspected that the abalone were invaded
by P. shioyasakiensis SDCH87 through the pedal tissues.
4.
DISCUSSION
Based
on bacterial isolation, sequence
analysis, and experimental
challenges in vitro and in vivo, we identifiedP.
shioyasakiensis strain SDCH87 as the causative agent of this outbreak.
To our knowledge, this is the first report of an outbreak associated
with P. shioyasakiensis causing high summer
mortality in abalone. Here, the
diseased abalone lacked external
lesions,
but the decrease in attachment abilities and the bleached pedal
epithelial cells suggested that the disorder was localized to the foot.P. shioyasakiensis was the
most abundant bacterial species isolated from the
moribund abalone. In addition,
eight strains were isolated from the mucus, four strains were isolated
from the foot, one strain was isolated from the gut, and one strain was
isolated from the digestive gland.
This distribution of strains may
also indicate that the foot was the primary site of P.
shioyasakiensis infection. Histological examination showed that the
structure of the pedal mucosa was damaged. Our experimental results
suggested that P. shioyasakiensis strain SDCH87 strongly
disrupted the first line of defense
(the mucosa) within three days of
exposure at 28 °C, and led to acute
mortality within a short time. Strain SDCH87 lost pathogenicity at 25
°C, indicating that higher temperatures allowed this strain to exhibit
opportunistic pathogenic behaviors. Furthermore, the 15 P.
shioyasakiensis strains were resistant to multiple antibiotics, and, of
all the chemotherapeutic agents tested, were only susceptible to
chloramphenicol. This may indicate
antibiotic misuse.
In
recent years, China has become the
largest producer of abalone globally, producing more than 90% of all
the farmed abalone worldwide (Cook, 2019). Due to its natural climatic
advantages, Fujian Province is the main abalone producing area in China
(Cook, 2019). However, the high-density aquaculture of the Pacific
abalone, which is the dominant cultured abalone species in China, has
led to outbreaks of various serious diseases, with
fatal consequences for the Chinese
abalone industry (Cook, 2019).
Several abalone diseases have previously been associated with various
pathogens (Bathige et al., 2016; Brevik et al., 2011; Fukui et al.,
2010; Hu et al., 2018; Kamaishi et al., 2010; Nicolas et al., 2002;
Nishimori et al., 1998; Sawabe et al., 2007), but it is unclear how the
causative pathogens were identified from among dozens of possible
contenders (Ren et al., 2019).
Here, 54 bacterial strains were
isolated and identified from
moribund abalone.
Vibrionaceae and
Pseudoalteromonadaceae were the two most abundant families in the
moribund abalone (41% and 23%, respectively). It was thus difficult to
test the pathogenicity of each strain. This difficulty was compounded by
the lack of visible lesions on the juvenile abalone.
The body mucus of most aquatic animals is highly resistant to
pathogen invasion, as this surface
is the interface between the host and external conditions, and is
directly immersed in the complex marine environment (Bakshani et al.,
2018). Symptoms of disease in
aquatic animals are usually accompanied by changes in the composition of
the microorganisms in the mucus on the body surface
(Vezzulli et al., 2018;
Zozaya-Valdes et al., 2015). We used Pseudoalteromonas, which
exhibited the greatest variation in abundance between the moribund and
health abalone, to test bacterial
mucus-resistance ability. After recording the mucus resistance of
various Pseudoalteromonas strains for 240 min, we postulated that
an increase in OD value after 120 min could not be used as an indicator
of bacterial resistance, because active substances in the mucus might
eventually break down into bacterially-available nutrients over time, as
was shown previously (Guo et al., 2009). Here, we identified
strain SDCH87 as pathogenic based
on bacterial growth data.
It
is well known the bacterial virulence factors can resist effector
molecules associated with host immunity, including metalloproteases
(Choudhury et al., 2015), extracellular proteases (Ridgway et al.,
2008), and collagenase (Bhattacharya, Bhattacharya, Gachhui, Hazra, &
Mukherjee, 2019).
Therefore,
bacterial resistance to mucus is
extremely important, and it can manifest as growth ability in
mucus (Fuochi et al., 2017;
Jung-Schroers et al., 2019; Wright et al., 2019).V.
alginolyticus strain Wz11, which had strong hemolytic activity,
exhibited high survival rates after a 2 h co-incubation with both
liquids of S. pharaonis compared withV. harveyi strain Wz21 and
caused higher mortality (Lv et al.,
2019). Consistent with the results
for V. alginolyticus strain
Wz11, the high pathogenicity of
strain SDCH87 was subsequently demonstrated according to Koch’s
Postulates. Critically, monitoring changes in microbial composition in
mucus is a rapid, non-invasive way to identify epidemic-causing
pathogens.
Here,
we demonstrated that our method effectively identified pathogenic
organisms; our results also provide a framework for the identification
of new pathogenic bacteria.
Species
of Pseudoalteromonas , one of the
most common bacterial genera in the
marine environment,
generally
act as
probiotics
in coral (Moree et al., 2014; Muchlissin, Sabdono, & Permata, 2018;
Rosado et al., 2019; Sabdono, Sawonua, Kartika, Amelia, & Radjasa,
2015; Shnit-Orland, Sivan, & Kushmaro, 2012), abalone (Offret, Jegou,
Mounier, Fleury, & Le Chevalier, 2019; Offret, Rochard, et al., 2019),
marine bivalves (Desriac et al., 2014; Rodrigues, Paillard, Dufour, &
Bazire, 2015; Sun et al., 2016) shrimp (Amoah et al., 2019; Pham et al.,
2014), lobster (Goulden, Hall, Pereg, Baillie, & Hoj, 2013), sea
cucumbers (Chi et al., 2014; Zheng
et al., 2012), fish (Mladineo et al., 2016; Sayes, Leyton, & Riquelme,
2016; Verner-Jeffreys, Shields, Bricknell, & Birkbeck, 2004), marine
algae (Albakosh, Naidoo, Kirby, & Bauer, 2016; Nagel et al., 2012), and
sea stars (Lloyd & Pespeni, 2018).
Only a few reports have identifiedPseudoalteromonas species as pathogenic to marine organisms,
including fish (Nelson & Ghiorse, 1999; Pujalte, Sitja-Bobadilla,
Macian, Alvarez-Pellitero, & Garay, 2007), crabs (Talpur et al., 2011),
algae (Goecke, Labes, Wiese, & Imhoff, 2013; Schroeder, Jaffer, &
Coyne, 2003), and sea cucumbers (Liu et al., 2010).
Our results indicate that SDCH87, a
strain of P.shioyasakiensis , is serious
pathogen. We isolated this strain from the pedal mucus of moribund
abalone. Strain
M14-00202-5E,
which showed 100% sequence identity with SDCH87,
was isolated in a
summertime mass mortality of
Pacific oysters in Cromartys Bay, Australia; in the affected oysters,
Pseudoalteromonadaceae was more abundant than in unaffected oyster
stocks (Go et al., 2017; King, Jenkins, Go, et al., 2019). However, the
pathogenicity of M14-00202-5E has yet to be clearly established, andVibrio species were considered as the probable causative
pathogens of oyster disease in this summertime mass mortality and
previous studies (Go et al., 2017; King, Jenkins, Go, et al., 2019).
Importantly,
this summertime Pacific oyster die-off coincided with high water
temperatures during the austral summer of 2014 (January 6–13) (Go et
al., 2017; King, Jenkins, Go, et al., 2019).
Similarly, a
record-breaking heat wave
in southern China in 2018 (K. Deng
et al., 2019; K. Q. Deng et al., 2020) was associated with the
high
abalone mortality investigated herein. Air
temperature data for Dongshan
country (http://www.tianqihoubao.com/lishi/dongshan.html) show that,
over the past three years, the period from March 2018 to June
2018
has been 2 ºC warmer than this same period during the preceding two
years. It rained on only 7 days
between late March to and April in 2018, as compared to 17 days in 2017
and 16 days in 2016. Due to the heat wave
in southern China, the average
minimum and maximum
air
temperatures increased to 25.6ºC and 32.3ºC, respectively, in late May
2018. Meanwhile, mean seawater
temperature
reached a record 28.5ºC, according to annual sea temperature records
from Daping Island, Dongshan Country (Fig. 1D).
Heatwaves are considered one of the
most destructive weather events associated with the changing climate (K.
Deng et al., 2019; K. Q. Deng et al., 2020). Recent heatwaves have
significantly impacted marine organisms and fisheries
worldwide (Caputi et al., 2016;
Cheung & Frolicher, 2020; Green et al., 2019; Lamb et al., 2018). In
conjunction, our analyses in vivo experiments at 25 °C and 28 °C
suggested that P. shioyasakiensis played an opportunistic role in
abalone mortalities due to the record-breaking heat wave in southern
China in 2018. Changing ocean temperatures have generally coincided with
rising rates of disease and mortality in many marine taxa (Caputi et
al., 2016; Cohen et al., 2018; Sanderson & Alexander, 2020), including
corals (Bally & Garrabou, 2007; Harvell et al., 2002; Porter et al.,
2001), marine gastropods (Fukui et
al., 2010), and oysters (Green et al., 2019; King, Jenkins, Seymour, &
Labbate, 2019). Acute mortality in rainbow trout during the summer
months was associated with an
emerging Lactococcus garvieae infection (Shahi, Mallik, Sahoo,
Chandra, & Singh, 2018), while the unexpected mortality of fan mussels
during summer 2017 was associated with Haplosporidium pinnae(Panarese et al., 2019). Finally, an
emerging summer pathogen of black
rockfish caused skin ulcer disease (Zhang et al., 2019). Elevated
temperatures may enhance the pathogenicity and adaptability of many
marine microorganisms by increasing metabolism and decreasing generation
time (Cohen et al., 2018; Hernroth & Baden, 2018). This may imply that,
with continuing climate change, more emerging
opportunistic pathogens will
challenge the health of aquatic organisms (Larsen et al., 2018). The
prevalence and high abundance ofP.
shioyasakiensis may represent a novel, important biomarker
of the health of cultured pacific
abalone, or even mollusks in general. Importantly, to promote the
responsible development of the aquaculture industry, we must consider
the complex interactions among host, pathogen, and environment that have
led to the recent global emergence
of opportunistic pathogens and increases in mass disease events.