Molecular Research in Fish Infectious Diseases

The Molecular Research in Thailand's Aquaculture
: Pathogen Characterization and

Antimicrobial resistance Monitoring
Assoc.Prof.Dr. Channarong Rodkhum
Center of Excellent in Fish Infectious Diseases
Department of Veterinary Microbiology
Faculty of Veterinary Science
Chulalongkorn University
Bangkok, THAILAND
• An estimated 73-180 billion fish are
farmed each year – making them
the most farmed vertebrate in the
world.
• Higher fish welfare means better
lives for billions of fish, on farms
YIELD FROM FRESHWATER AQUACULTURE BY
SPECIES (Department of Fisheries, Thailand)
World production of Tilapia
4,500,000 tons/year
The most consumed Fish globally & it is
now called
AQUATIC CHICKEN
SOURCE: FAO
Successful and sustainable in fish farming come from
successful in fish health managements
Health Booster
(Probiotics,
immunostimutants,
vaccines)
Farm, water
quality and
Nutritions environmental
management
Successful
in fish
farming
Breed
farming skills
and Basic
(diseases
Knowledges resistance
Fish)
Biosecurity
5
Fish health management
• Appropriate stock density
• Appropriate nutrition for each breed and

period
• Water qualtity management and

monitoring
• Supplement with immunostimulants such as

vitamins, trace minerals and betaglucan
• Use Probiotics or Synbiotics
• Vaccination for important diseases
6
Failure of fish health management
Diseases
7
Important infectious diseases
of Tilapia
• Streptococcosis
• Columnaris
• Haemorrhagic septicemia such as Aermonas septicemia

and Edwardsiella septicemia
• Franciscellosis
• Tilapia Tilapinevirus infection
• Parasitic infection such as Trichodina and Dactylogyrus
• Mixed infection or concurrent infections
8
Tilapia major diseases
Major diseases affecting tilapia

during the farming cycle
9 Source : http://www.thefishsite.com/articles/139/ diseases-of-tilapia-an-introduction

Streptococcosis
10
Columnaris disease
11
Bacterial hemorrhagic septicemia
12
Franciscellosis
Tilapia Tilapinevirus infection
14
Important diseases of Sea Bass
Marine culture
• Streptococcosis
• Taenecibaculosis
• Scale drop and muscle necrosis from Vibrio spp. (Vibriosis)
• Scale drop disease virus infection
• Infectious spleen and kidney necrosis virus (ISKNDV) infecttion
• Viral Nervous Necrosis (VNN) virus infection
• Iridovirus infection
• Parasite
Freshwater culture
• Streptococcosis
• Columnaris
• Aeromonas septicemia
Diseased fish usually infected by multiple organisms called

concurrent infection.
15
Diseases of Sea Bass
Hatchery Nursery Pre-grow out Grow out

Day
0 gram 1 gram 15 gram 100 gram 1.5 Kg
Emerging diseases
Complied from : https://thefishsite.com/ articles/ diseases-of-farmed-barramundi-in-asia

16
Columnaris in Freshwater culture Sea Bass
17
Taenecibaculosis in marine culture Sea Bass
18
Scale Drop Disease virus (SDDV) infection
19 https://doi.org/10.1111/jfd.12915
Bacterial Scale Drop and Muscle Necrosis disease
(SDMND)
20 https://aquahealth.wordpress.com
Infectious Spleen and Kidney Necrosis virus (ISKNDV) infection
21 https://aquahealth.wordpress.com
Viral Nervous Necrosis (VNN)
22
https://thefishsite.com/articles/diseases-of-farmed-barramundi-in-asia
Iridovirus infection
23
Streptococcosis
24
Molecular research workflow for aquatic animal
pathogens and their antimicrobial resistance
Disease investigation Molecular techniques and

other advanced
techniques
❑ PCR
❑ Real time PCR
Necropsy and Sample collection ❑ Sequencing
❑ Fresh ❑ Phylogenetic analysis
❑ Absolute alcohol fixation ❑ Bioinformatics
❑ Formaline fixation ❑ LAMP
❑ RPA
❑ VITEK II system
❑ MALDI-TOF
Isolation and Identification

the pathogens
❑ Conventional techniques
❑ Immunological techniques
❑ Molecular techniques
25
CASE STUDY ON FLAVOBACTERIUM INFECTION
Assoc. Prof. Dr. Channarong Rodkhum

Center of Excellent in Fish Infectious Diseases (CE FID)
Department of Veterinary Microbiology
Faculty of Veterinary Science, Chulalongkorn University,
THAILAND
INTRODUCTION
 Most disease laboratory studies are typically aimed at single

infection.
 Fish is naturally exposed to various potential pathogens in
water
 Hypothesis: The reality of dead-loss in cultured fish farms
due to multiple infections probably outweighs single
infection.
OUTBREAKS IN NONG KHAI
August, 2014 (1)
 Occur annually in floating

cage cultured Nile tilapia
farms along the Mekong River
 Mortality: ~ 50%
 Clinical signs
➢ External – columnaris-like
➢ Internal – hemorrhage
 Sample collection:
10 moribund from 2 affected
farms (5 each)
OUTBREAKS IN NONG KHAI (2)
Methodology
 Bacterial isolation: AOA (F. columnare) , CHA (Francisella) , TSA
(other).
 Phenotypic & 16S rRNA sequencing for pure bactetial isolates
 Specific PCR diagnosis for internal organs of diseased fish:

Franciscella sp., Iridovirus, F. columnare.
Result of bacterial isolation and identification (1)
16S rRNA
Biochemical Genbank Identity
Isolates Most closely related species
profile accession (%)
number
NK-Fc01, NK-Fc02,
NK-Fc03, NK-Fc04,
NK-Fc05, NK-Fc06,
KP899505- Flavobacterium columnare
ND 100
KP899514 CUVET1201(KF274033)
NK-Fc07, NK-Fc08,
NK-Fc09, NK-Fc10
Aeromonas veronii bv. veronii
NK01, NK03, NK04 1 99.9
KP899499- strain ATCC 35624 (NR118947)
NK02, NK05, NK06, KP899504 Aeromonas veronii bv. veronii
2 99.9
NK07 strain ATCC 35624 (NR118947)
Vibrio cholerae strain PIM9
NK08, NK12 3 KP899517 99.6
(GQ359963)
KP899515- Plesiomonas shigelloides strain
NK10, NK11 4 99.8
KP899516 NCIMB 9242 (NR044827)
KP899518- Streptococcus agalactiae
NK09, NK13, NK14 5 99.9
KP899519 strain ATCC 13813 (NR115728)
Result of bacterial isolation
and identification (2) A. veronii
P. shigelloides
V. cholerae
S. agalactiae
F. columnare
Result of specific PCR diagnosis
Concurrent infection of bacteria and virus
found in two affected tilapia farms.
F. A. S. Francisella P. V. Iridovirus Number of
columnar veronii agalactia spp. shigeloides cholerae pathogens
per fish
e e
Farm 1 (%) 100 100 0 0 0 40 40

Fish 1 + + - - - - + 3
Fish 2 + + - - - - + 3
Fish 3 + + - - - + - 3
Fish 4 + + - - - - - 2
Fish 5 + + - - - + - 3
Farm 2 (%) 100 100 60 0 20 0 20
Fish 1 + + + - - - + 4
Fish 2 + + + - - - - 3
Fish 3 + + - - + - - 3
Fish 4 + + + - - - - 3
Fish 5 + + - - - - - 2
Experimental challenge (1)
Treatment Number Bacteria administered Infection Challenge dose
group of fish route
1 10 A. veronii NK01 I.P. injection 1.36 × 107 CFUs fish-1
2 10 A. veronii NK06 I.P. injection 0.82 × 107 CFUs fish-1
3 10 S. agalactiae NK13 I.P. injection 2.30 × 108 CFUs fish-1
4 10 P. shigeloides NK11 I.P. injection 1 × 107 CFUs fish-1
5 10 F. columnare NK-Fc01 I.M. injection 1 × 107 CFUs fish-1
6 10 TSB (Control) I.P. injection 0.1 mL sterileTSB
CFUs, colony forming units; I.P., intraperitoneal; I.M., intramuscular; TSB, tryptic soy broth
Red tilapia fingerlings model

Naturally diseased fish Experimentally diseased fish
F. columnare
A. veronii
Other evidence of concurrent infection
during disease outbreaks (1)
▪ Francisella noatunensis subsp. orientalis +

Flavobacterium columnare
Conclusion
 The reality of dead-loss in cultured fish farms due to multiple
pathogen infections outweighs single infection
Other evidence of concurrent infection during disease outbreaks
Naturally Experimentally
diseased fish diseased fish
Virulence and Characterization of
Flavobacterium columnare from Diseased Tilapia
Conceptual Framework
Isolation and identification isolates of Flavobacterium

columnare from red tilapia (Oreochromis spp.) in Thailand
F. columnare isolates
Phenotypic classification Genotypic classification
Summary of the characteristics of Flavobacterium columnare isolated

from red tilapia (Oreochromis spp.) in Thailand
41
BACTERIAL ISOLATION & IDENTIFICATION (1)
Geographical locations for sample collection

Bacterial isolation (AOA)
49 isolates
Red tilapia: n=44
Nile tilapia: n=1
Striped catfish: n=3
42 Koi carp: n=1 Phenotypic tests (Bernadet, 1989) Specific PCR (Welker et al., 2005)
GENETIC CHARACTERIZATION OF F. COLUMNARE (1)
➢ 16S-RFLP (n=49) (Darwish et al., 2005; Lafrentz et al., 2014)
➢ ISR Sequencing & Phylogenetic analysis (n=25) (Zavaleta et al., 1999)
➢ 16S RNA sequencing & Phylogenetic analysis (n=19) (Darwish et al., 2005)
43
GENETIC CHARACTERIZATION OF F. COLUMNARE (2)
Genomovar assignment (16S-RFLP)

• 20F& R1500 primers → failed
• 20F& R1438 primers → success → HaeIII digestion
II I
M
~ 600 bp
500 bp ~ 430 bp
300 bp ~ 290 bp
~ 276 bp
200 bp ~ 180 bp
100 bp
Genomovar II: 48 isolates

Genomovar I: 1 isolates (CUVET1215)
44
45
Unique cluster
GENETIC CHARACTERIZATION (3)
50
G Thai isolates (25)
Branch II
83
F. columnare BZ-04 Branch II
99 F. columnare BZ-01 F
97
99 F. columnare BZ-02
E
CUVET seq (n=34)
F. columnare BZ-05
99 F. columnare CUVET1231
100
GenBank seq (n=16) F. columnare CUVET1232 D
96 F. columnare ALG-00-530
Thai isolates formed 3 clusters 81 F. columnare EK 28

64 F. columnare LP 8
ISR-2 (587-651 bp)- Branch I (n=9) 99 F. columnare LV-339-01

F. columnare CUVET1360
ISR-1 (523 -537 bp)- Branch II (n=25) 98
F. columnare CUVET1363 C
96
Genomovars & Clusters 68 F. columnare CUVET1368
F. columnare CUVET1372 Branch
Branch I I
100 F. columnare ATCC 49513
F. columnare ATCC 23463
B
99 F. columnare FK 401
77 100 F. columnare ATCC 49512
56 F. columnare ARS-1
100 F. columnare PH 97028
100 F. columnare AU-98-24 A
F. columnare GA-02-14
94
Flavobacterium johnsoniae ATCC 17061
Flavobacterium psychrophilum ATCC 49418
46
0.05 Phylogenetic tree based on ISR of F. columnare isolates
Unique cluster
F. columnare CUVET1348 18 isolates
F. columnare CUVET-BU
64
7 isolates 100
47
0.005
50
G
83
F. columnare BZ-04 Branch II
99 F. columnare BZ-01 F
97
99 F. columnare BZ-02
F. columnare BZ-05 E
100 F. columnare CUVET1232 D
96 F. columnare ALG-00-530
81 F. columnare EK 28
64 F. columnare LP 8
99 F. columnare LV-339-01 16S rDNA sequencing

F. columnare CUVET1360 n=19
F. columnare CUVET1363 C
98
96
F. columnare CUVET1372 Branch I
B
99 F. columnare FK 401
77 100 F. columnare ATCC 49512
56 F. columnare ARS-1
100 F. columnare PH 97028
100 F. columnare AU-98-24 A
F. columnare GA-02-14
94
Flavobacterium psychrophilum ATCC 49418
48
Phylogenetic tree based on ISR of F. columnare isolates
0.05
GENETIC CHARACTERIZATION (6) F. columnare CUVET1243
• CUVET seq (n=19) F. columnare CUVET1241

•
GenBank seq (n=17) 99 F. columnare CUVET1338 Sub-cluster
Sub-cluster IIB
• 1180 bp (position 90-1278 E. coli
F. columnare CUVET1214 IIB
numbering) F. columnare CUVET1212
Cluster
Cluster II
• Unique cluster ISR-G (n=16) 54
F. columnare CUVET1202 II
formed a sub-cluster IIB F. columnare CUVET1201
F. columnare LP8
72
F. columnare EK28 Sub-cluster
F. columnare LV339-01
90 F. columnare Ga-6-93 IIA
98 Sub-cluster IIA
F. columnare ALG-00-530 clone GII-18
F. columnare PT-14-00-151 clone GIIB-1
Genomovar II-B → 61
99 57 F. columnare ATCC 23463 clone GI-1
Genomovar I/II →
99
F. columnare F10-HK-A clone GI/II-1
F. columnare IAM 14301 Cluster I
Cluster I
98
F. columnare FK 401
99
F. columnare AU-98-24 Cluster
Genomovar I (CUVET1215) →
F. columnare PH-97028 Cluster III
86
97 F. columnare GA-02-14 clone GIII-1 III
52 F. columnare ARS-1 clone GIA-1
Flavobacterium psychrophilum strain ATCC 49418
75 Flavobacterium branchiophilum NBRC 15030.
49 0.03 0.02 0.01 0.00

DISCUSSION
Good agreement between ISR and 16S rDNA phylogenetic

I
analysis
ISR phylogenetic 16S rDNA phylogenetic

tree tree
Unique cluster (ISR-G) Unique sub-cluster (IIB)
16S-RFLP result is not correlated to 16S-rDNA phylogenetic

II
analysis
16S rDNA
16S-RFLP phylogenetic tree
Genomovar I/II (F10-HK-A) Genomovar I (Cluster I)
Genomovar II-B (PT-14-00-151) Genomovar II (Sub-cluster IIA)
Genomovar I (CUVET1215) Genomovar III → Cluster III
50
VIRULENCE OF RHIZOID AND NON-RHIZOID
MORPHOTYPES (1)
CUVET1214 CUVET1201
 Upon primary isolation, both isolates formed yellow-
pigmented rhizoid colonies on AO agar
 CUVET1201 transformed to non-rhizoid under the
preservation condition in the laboratory
51
MORPHOTYPES (2)
Virulence assay Adherence and persistence assay
Red tilapia fry (~0.39 g)

52
MORPHOTYPES (3)
Virulence assay
Naturally Experimentally
diseased fish diseased fish
53
MORPHOTYPES (4)
Adhesion & Persistence
The results suggested that an inability of the non-rhizoid

morphotype to persist in tilapia fry may explain lack of
virulence.
54
55
CASE STUDY ON
STREPTOCOCCOSIS
IN AQUACULTURE
56
STREPTOCOCCOSIS IN FISH
CAUSATIVE AGENTS
• Non-motile, non spore-forming and Gram-
positivestreptococcus related species
– Streptococcus agalactiae (Group B
streptococci)
– S. iniae
– Lactococcus garvieae
– S. dysgalactiae, S. parauberis, etc.
• Produce either complete- (β), incomplete-
(α) haemolysis
57
•GRAM POSITIVE,
ENCAPSULATED,
COCCI, NON-SPORE
FORMING AND MOSTLY
ß-HEMOLYSIS
• WIDE HOST-RANGE
PATHOGEN
58
STREPTOCOCCOSIS IN FISH
• Causing acute mortality in variety of

fish
– Tilapia, Asian seabass, salmon,
trout, pangasius
– Marine mammal (Evans, 2006)
• >50% mortality could be found within
1-2 weeks
• Mainly transmitted via horizontal
transfer
– But vertical transfer is likely feasible
(Amal, 2013)
• Zoonosis and reverse-zoonosis are
capable (Yanong, 2007)
59
60
• Increasing of water temperature can significantly exacerbate
disease virulence
– Promote bacterial growth and expression of virulence

factors (capsule, haemolysin, pilus, etc.)
– Host response → massive inflammation leads to acute
mortality
10 100
Acuumulated percent mortality

CFU/g brain (log10)
8 80
6 60
4 40
2 20
0 0
0 6 12 24 48 72 96 1 2 3 4 5 6 7 8 9 10 11 12 13 14
time (hpi) day post infection (dpi)
control 28 ⁰C control 35 ⁰C control 28 ⁰C control 35 ⁰C

challenge 28 ⁰C challenge 35 ⁰C challenge 28 ⁰C challenge 35 ⁰C
Number of viable bacteria resided in brain Accumulated mortality of infected tilapia

MAJOR STREPTOCOCCUS SP.
IN THAILAND
S. agalactiae
(Group B Streptococci)
S. iniae
L. garvieae (FROM
PRAWN)
62
VARIABILITY OF S. agalactiae IN
THAILAND
SUB-SPECIES
CATEGORIZATION
• Serotyping (Ia, Ib, II-IX)
– Human: Ia, III, V
– Bovine: non-specific
serotype
– Fish: Ia, Ib, III
• Cross-protection of
heterologous serotype is
very limited (Cieslewicz et al., 2005)
DISTRIBUTION OF S. agalactiae SEROTYPE
CHINA &
NORTH
CENTRAL ASIA
AMERICA
Ia Ia, Ib
EUROPE
Ia SEA
Ia, Ib, III
SOUTH
AMERICA
Ib AUSTRALIA
Ia
64
Most were identified as molecular
serotype Ia (only 2 isolates were type III)
• Serotype Ia was the majority of the
fish originated S. agalactiae in
Thailand (Suanyuk et al., 2008)
High genetic variation among S.

agalactiae
• Geographically dependent
• Vaccine application might be hardly
imply 65
Antimicrobial susceptibility of S. agalactiae
• Streptococcal bacteria are susceptible

to amoxicillin, ﬂorfenicol,
sulfamethoxazole/trimethoprim and
sulfadimethoxine/ormetoprim
• However, enrofloxacin-resistant
strains were emerged over the recent
years
(Lukkana, 2015)
66
CONCLUSION
• At least 3 species of Streptococcosis associated

bacteria have circulated in Thailand
• Streptococcus agalactiae type Ia is major cause of
streptococcosis in Thailand
• High temperature (>32 °C) is a risk factor
– Massive inflammation related to mortality of fish
• High genetic variation with geographically
dependent
• Universally efficient vaccine is awaits to be
developed
67
DISTRIBUTION OF S. agalactiae SEROTYPE
(Personal communication)
SEROTYPE Ia,
III (highly pathogenic)
(Kayansamruaj, 2014)
SEROTYPE Ia
(Suanyuk, 2008)
SEROTYPE Ia,
III (human originated
strain)
68
Research Outcome
69
Research outcome
70
Research Outcome
71
Case study of Franciscellosis in
Tilapia
Distribution of Franciscellosis in fish
• Ricketsia-like organism (Chen et al. 1994)
• Piscirickettsiosis-like syndrome (Mauel & Miller 2002)
• Francisella noatunensis subsp. orientalis in warm water fish
F. noatunensis subsp. noatunensis in cold water fish

(Mikalsen and Colquhoun, 2009; Ottem et al., 2009)
76
Manuel, 2010
Fno -> francisellosis
Media Optimum
Cystine heart agar + 5 % sheep blood Growth condition
temperature:
aerobic
(Soto et al., 2009) 25-28 °c
• Culture fish pathogenic Francisella is complicated
• Highly fastidious in growth requirements
• Easily inhibited by other bacteria
• Concurrent antibiotic treatment

77
Bacterial isolation and identification
- PCR
- Histology (H&E,
Giemsa, Gram stain)
- Bacterial isolation
(CHA, TSA)
Freshly dead fish

Host: red
•Colonies: tilapia
grey, (n = 10)
convex, mucoid Gram (-)Suspected
Collection date:
•Morphology: September 2013Catalase:colonies
cocco-bacilli (+) Oxidase: (-)
• Geographical origin: Kanchanaburi 9
Bacterial isolation and identification
DNA
Primers extraction
Sequence (5’-3’) Target gens References
F11 TACCAGTTGGAAACGACTGT 16S rRNA of Forsman et al.,
• Bacterial
F5
colony sample: boilingFrancisella.
CCTTTTTGAGTTTCGCTCC
methodsp (Shah et al.,
19942012)
F1 GAGTTTGATCCTGGCTCAG 16S rRNA
• Fish tissue sample (spleen, kidney):(Universal
DNeasy bloodDorsch
& tissue
&
R13 AGAAAGGAGGTGATCCAGCC Stackebrandt 1992
kit (Qiagen) primer)
10
Data analysis
• Sequencing analysis
• CLUSTALX 2.0
• BLASTN program.
• MEGA10
• One-way ANOVA to test difference between each experimental
groups
79
RESULTS
Bacterial identification
M: 1 kb ladder
1 kb
1-2: Healthy fish
3-12: Affected fish
Figure 1: PCR diagnosis of Francisella sp. from
affected red tilapia using genus-specific primers.
• One random PCR product: 1009 bp sequence

• GenBank: accession number KJ92505
• 100% sequence identity with published sequences
of Francisella noatunensis subsp. orientalis
80
RESULTS
A B
Figure : Fno VMCU-FNO 131 on CHA (A) and Gram staining (B)
• 16S rRNA gene:

M: 11393 bp sequence
kb ladder
• GenBank: accession number
1: Francisella genusKJ841395
specific
primer
• 100% sequence identity with published sequences
Figure 2: universal
5: PCR amplification primer.
of strain VMCU-FNO131
16 of Francisella noatunensis subsp. orientalis
79
RESULTS
Francisella noatunensis subsp. orientalis strain Ehime-1 NR_112536 threeline grunt Japan
F. noatunensis subsp. orientalis strain CUVM-FNO131 KJ841935 tilapia Thailand
100
F. noatunensis subsp. orientalis AF-04-405 DQ007455 tilapia Taiwan
I
F. noatunensis subsp. orientalis strain Ind04 FJ217163 tilapia Indonesia
76
F. noatunensis subsp. orientalis LADL 07-285A EU672884 tilapia Costa Rica
F. noatunensis sbsp. noatunensis AM403242 Atlantic salmon Chile
F. noatunensis subsp. noatunensis NR_043696 Atlantic cod Norway
100 II
F. noatunensis subsp. noatunensis EF490217 Atlantic cod Norway
67
F. noatunensis subsp. noatunensis EF685354 Atlantic cod Norway
F. halioticida AB449247 giant abalone Japan
F. tularensis subsp. tularensis AY968225 environmental sample
0.002
Figure Phylogenetic tree was constructed based on alignment of 1380bp

of 16S rRNA sequences from Francisella species
82
RESULTS
Experimental challenge to fulfill Koch’s postulates
Natural infected Experimental fish
Control fish
21
RESULTS
A B
Figure : . A) Granulomas formation (G) in infected red tilapia
and granuloma in high magnificent. B) Normal tissue from
control fish.
84
Histological lesions of red tilapia affected by Fno
K
S
A B
L I
D C
85
Typical granuloma caused by Fno infected in red tilapia
Early
stage
20 µm A B 20 µm
Last Mature
stage stage
20 µm
D C 20 µm
24
Histological lesions of control red tilapia
K
S
A B
L I
D C 25
RESULTS
A B
Figure 9: A) Primary and secondary gill lamella of infected red tilapia
presented hyperplasia and fusion, and granulomas inflammation at basal of
gill arch. B) Normal gill of control fish. H&E stain.
26
RESULTS
Recovered
isolates
(B)
Challenge
red tilapia
(A)
Figure 10: A) Lanes M: DNA marker; 1-8: challenge fish; P:

positive control; N: control fish. B) Lanes: M: DNA marker;
1-12: Fno isolates; P: positive control; N: negative control
27
RESULTS
100
90
Cumulative mortality %
80
70
60
2.88
Dose 1x 105 CFU per fish
50
2.88
Dose 2x 103 CFU per fish
40 Control
Control
30
20
10
0
1 3 5 7 9 11 13 15 17 19
Days after challenge
Figure : Cumulative mortality of red tilapia challenged by VMCU-FNO131 via i.p
28
RESULTS
Virulence assay of VMCU-FNO131 in red tilapia
• Clinical signs
• White nodules were only observed in dose 105,
104, 103 CFU fish-1 via i.p
• Other doses: enlarged internal organs
• West mount and histological finding in both i.p
and immersion route were common with previous
experimental challenge
29
RESULTS
100
90
7Dose
x 10
1 6 CFU mL-1
80
70 2.88
Dose 2 x 105 CFU mL-1
60
2.88
50
40
2.88
30 Control
Control
20
10
0
Day
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Figure: Cumulative mortality of red tilapia via immersion route
30
RESULTS

100
90
80
70 2.881x
Dose 105 CFU per fish
60 2.882x
Dose 104 CFU per fish
Dose 3
50 2.88 x 103 CFU per fish
Dose 4
40 2.88 x 102 CFU per fish
COntrol
30 Control
20
10
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Day
Figure : Cumulative mortality of red tilapia via i.p route

31
RESULTS
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
2 kb
Challenged
1 kb
red tilapia
(A)
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 P N
1 kb Recovered
(B) isolates
Figure 15: A) 1-10: infected fish in injection group; 11-21: infected fish in
immersion group; P: positive control; N: DNA extracted from control fish.
B) M: DNA marker; 1-14: isolates recovered from injection groups; 15-17:
isolates recovered from dose 105 & 106 CFU mL-1 of immersion groups.
33
Table : Summary of Francisella noatunensis subsp. orientalis
VMCU-FNO131 virulence assay in red tilapia
Number of Number of PCR test Mean number of granulomas

Cumulative
isolates isolates for in 10X microscopic field
Trial dose mortality
recovered from recovered from survivors Head
(%) Spleen Liver
dead fish survivors (%) kidney
Intraperitoneal injection (CFU per fish)
2.88 x 105 90 3/4 1/1 100 Severe Severe Mild
2.88 x 104 80 3/4 2/2 100 Severe Severe Mild
2.88 x 10 3 30 2/3 2/4 100 Moderate Severe Mild
2.88 x 102 30 2/3 - 100 Moderate Moderate Mild
Control 0 N/A - - N N N
Immersion (CFU/ml of tank water)
7 x 106 25 1/2 3/5 100 Moderate Severe Mild
2.88 x 105 0 N/A 2/4 100 Moderate Severe Mild
2.88 x 10 4 0 N/A - 100 Moderate Moderate Mild
2.88 x 103 0 N/A - 100 Moderate Moderate Mild
Control 0 N/A - - N N N
Notes:
a) Positive = + b) Negative = - c) N/A = Not Applicable
d) Severe = X > 20 e) Moderate = 7 < X < 20 f) Mild = X < 7
95
Research Outcome
94
95
CASE STUDY :
Genome Characterization of Quinolone Resistance
Associated Genes of Flavobacterium columnare
Isolated from Columnaris Diseased Asian Sea Bass
(Lates calcarifer)
Putita Chokmangmeepisarn
Advisor : Asst. Prof. Dr. Channarong Rodkhum
Co-advisor : Dr. Pattanapon Kayansamruaj
Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University

IMPORTANCE & RATIONAL
Diseases of Asian sea bass
in Thailand
Viral Nervous Necrosis (VNN)
Scale drop disease virus (SDDV)

Streptococcus iniae
96 Vibrio sp.
▪ Gram negative long rod

bacteria
▪ Family Flavobacteriaceae
▪ Cultured media : Cytophaga
media
▪ Causative agent of columnaris
disease
▪ Optimum growth temperature for

outbreak : 20-30°C
▪ The bacteria exhibit gliding motility
▪ Colony morphology is yellow, flat,
rhizoid and sticky adhere to agar
97
(Bernardet and Bowman, 2006)
Columnaris disease
▪ A highly contagious disease
causing by bacteria
“Flavobacterium columnare“
▪ The pathogen commonly affect to
gills, skin, and fins
▪ yellow-grey opalescent necrotic
patches or erosions are visible on
the skin
▪ Severe tissue necrosis develops
into “saddleback” lesions on the
dorsum
▪ The lack of commercial vaccine
promote the use of antibiotics to
prevent and control the disease
98 (Durborow et al, 1998)
(Dong, 2014)
Antibiotic use
Imprudent use of antibiotics can be a public health concern
The human health hazard

associate with antimicrobial use
(AMU) in aquaculture
▪ Drug residues accumulation
in aquaculture products and
dissemination in environment
▪ Risen of antimicrobial
resistance (AMR)
▪ Resistance genes transfer
99
Antibiotic use
A several kind of antibiotics have been

permitted for use in Thai aquaculture
✓ Amoxicillin
✓ Enrofloxacin
✓ Oxytetracycline Quinolones are restricted or
✓ Sarafloxacin
✓ Oxolinic acid prohibited in some countries
✓ Toltrazuril because of the critical
✓ Sulfamonomethoxine Sodium importance of this class of
✓ Sulfadiazine + trimethoprim antibiotics
✓ Sulfadimethoxine sodium + trimethoprim
✓ Sulfadimethoxine sodium + ormethoprim for treating human pathogens
✓ Sulfamonomethoxine + trimethoprim
✓ Sulfadimidine + trimethoprim (WHO, 2007)
(DOF, 2012)
100
Antimicrobial
susceptibility tests
▪ There are 2 most common methods for testing

phenotypic antimicrobial susceptibility
Disk diffusion method Limitation
▪ F. columnare are fastidious
bacteria
▪ Usually clumping
▪ The protocol quite
complicated
Broth/agar dilution method ▪ Need more standardized
method
▪ Need the official breakpoint
criteria
101
(CLSI, 2014; Gieseker et al., 2016)
Detection of AMR genes
➢ AMR genes identification by PCR & DNA

sequencing
▪ Mutations associated with quinolone- Limitations

resistance phenotype were determined ▪ A single or few mediating resistance
▪ The first study that reveal the QR strains genes are tested
harbored missense mutations within parC ▪ Expensive and time-consuming to
and gyrA but not in gyrB or parE perform a complete identification of
resistance genes
▪ Some novel genes can be missed
(Mata et al., 2017) 102
➢ AMR genes identification by Next-generation sequencing (NGS)
▪ High-throughput sequencing
▪ Rapid
▪ Become more feasible
▪ Bioinformatics tools and free-access
databases are available
Advantages
▪ Predicted the antimicrobial
resistance and associated
genes
▪ Tracking a multi-drug
resistance mechanism
▪ Investigated horizontal gene
transfer
▪ Identify novel resistance gene
(Zankari et al., 2012)
103
➢ AMR genes identification by Next-generation sequencing (NGS)
▪ 286 genes were predicted

as AR genes blasted to
CARD database
▪ 32 genes were annotated
as RND-type efflux pumps
▪ Aminoglycoside and
kanamycin resistance
genes were identified
The systematical researches of quinolone resistance

mechanisms of F. columnare still unavailable
104
(Zhang et al., 2017)
MATERIALS & METHODS
Research Plan
F. columnare isolates Bacterial identification

▪ Phenotypic
▪ Molecular
Antimicrobial susceptibility tests

DNA extraction &
▪ Disk diffusion
Whole genome sequencing
▪ Broth microdilution
Resistome analysis
105
MATERIALS & METHODS
Sample collection &
Bacterial isolation
Sample colllection
▪ All isolates of F. columnare were isolated from

freshwater culturing Asian sea bass
▪ 2 farms in 2 provinces of Thailand; Chachoengsao Samutprakarn
and Samutprakarn
▪ Growth condition : culture with Anacker and Ordal
(AO) media and incubated at 28 °C for 48 hrs Chachoengsao
106
106
MATERIALS & METHODS
Sample collection &
Bacterial isolation
▪ Phenotypic identification : Gram staining & Biochemical tests

- Growth capacity on TSA and MacConkey agar
- Flexirubin pigment production
- Motility test
- Decarboxylase test
▪ Molecular identification : PCR & sequencing

Species-specific primers for 16s rRNA gene :
- FCISRFL (5'-TGCGGCTGGATCACCTCCTTTCTAGAGACA-3‘)
- FCISRR1 (5'-TAATYRCTAAAGATGTTCTTTCTACTTGTTTG-3‘)
- Product size : 400-500 bp
(Welker et al., 2004)
107
107
MATERIALS & METHODS
Antimicrobial
susceptibility tests Disk diffusion method
▪ 7 discs containing antimicrobial agents

were used
• Oxytetracycline
• Amoxicillin
• Florfenicol
• Oxolinic acid
• Nalidixic acid
• Enrofloxacin
• Ciprofloxacin
▪ The test were performed by using 1:5 diluted Mueller-Hilton

agar (DMHA)
▪ Inhibition zone diameter were measured
(CLSI et al., 2014) 108

MATERIALS & METHODS
Antimicrobial
susceptibility tests Broth microdilution method
▪ 3 kinds of quinolone & fluoroquinolone were used

• Oxolinic acid
• Nalidixic acid
• Enrofloxacin
▪ The antimicrobial stock (10240 μg/mL) will be prepared
and two-fold diluted into 10 concentration
F. columnare 0.5 McFarland 1:100 dilution in MIC were

Shaking at 28°C 160 rpm 48 hrs standard CAMHB determined
(CLSI, 2014; Gieseker et al., 2016)

109
MATERIALS & METHODS
DNA extraction
& Sequencing
▪ DNA were extracted by PromegaTM WizardTM Genomic

DNA purification kits
▪ DNA sequencing will be performed using Illumina® Hiseq
platform with 151 paired-end run length
110
MATERIALS & METHODS
Genome analysis
workflow
de novo assembly
Raw reads SPAdes Contigs

genome assembler
Antibiotic resistance genes prediction
Annotation
Rapid Annotation using
The Comprehensive Antibiotic Resistance Database Subsystem Technology (RAST)
Chromosomal mutations prediction
111
Resfinder
RESULTS & DISCUSSION
Sample collection
& bacterial isolation
▪ All fishes showed a A B

clinical signs of
columnaris disease
including gill necrosis
and fin erosion
▪ one fish was showed FID RU
saddle back lesion
C
FID RU
Asian sea bass with fin and tail rod (A),

gill necrosis (B), and saddleback lesion (C)
112
Sample collection
& bacterial isolation F. columnare isolates used in this study
Year
No. Isolates Region Organ
isolated
1 CC1801 Chachoengsao Skin 2018
▪ Total 15 isolates were
recovered from diseased Asian
sea bass 3 CC1803 Chachoengsao Skin 2018
▪ 8 isolates from Chachoengsao 4 CC1804 Chachoengsao Skin 2018
▪ 7 isolates from Samutprakarn 5 CC1805 Chachoengsao Skin 2018

▪ Flat, yellow, and rhizoid colony
on AOA 7 CC1807 Chachoengsao Skin 2018
9 SP1801 Samutprakarn Gill 2019
11 SP1803 Samutprakarn Skin 2019
FID RU 13 SP1806 Samutprakarn Gill 2019

113
Colony morphology
Phenotypic identification Characteristics Results
Gram Negative
Morphology Slender long rod
Growth on TSA -
Growth on MAC -
Flexirubin pigment +
Catalase +
Cytochrome oxidase +
FID RU Gliding motility +
Decarboxylase test
Gram staining Arginine -
Lysine -
Ornithine -
114
Molecular identification
PCR amplification of F. columnare isolates by

using species-specific primers
M N 1 2 3 4 5 6 7 8 9 101112131415
▪ All isolates were

obtained 400 – 500
500 bp
400 bp bp PCR product
▪ Sequencing results
were 90 – 100%
identity to
F. columnare
Lane M: DNA marker, Lane N: negative control, Lane 1-15: SP1801,

SP1802,
115SP1803, SP1805, SP1806, SP1808, SP1809, CC1801, CC1802,
CC1803, CC1804, CC1805, CC1806, CC1807, and CC1808 respectively.
Antimicrobial susceptibility
Disk diffusion
Antimicrobial susceptibility results from

disk diffusion method
No. of isolates
20
15
10
0
Antibiotics
OA NA CIP ENR OT AML FFC
Resistant
▪ 33.33% of isolates were resistant to oxolinic acid

▪ 40% of isolates were resistant to nalidixic acid
116
Disk diffusion
Antibiotics
Isolates
OA NA ENR CIP OT AML FFC
CC1801 S S S S S S S
CC1805 S S S S S S S Quinolone
CC1806 S S S S S S S Sensitive
CC1807 S S S S S S S (QS)
CC1808 S S S S S S S group
SP1801 R R S S S S S
Quinolone
SP1805 S R S S S S S
Resistance (QR)
group
SP1808 S S S S S S S
117
Broth microdilution
MIC (μg/mL)
Isolates
OA NA ENR
CC1801 0.25 2 <0.125
CC1802 0.125 2 <0.125
CC1803 0.25 0.25 <0.125
CC1804 0.25 0.25 <0.125
CC1805 0.25 4 <0.125
CC1806 0.25 4 <0.125
CC1807 0.25 4 <0.125
CC1808 8 <0.125 0.25
SP1801 32 >64 1
SP1802 32 >64 1
SP1803 8 >64 1
SP1805
SP1806
0.5
8
>64
>64
0.25
1
▪ 7 isolates were
118
SP1808 0.5 >64 0.5 selected for NGS
SP1809 16 >64 1
Genome features
CC1802 CC1803 CC1805 CC1808 SP1802 SP1805 SP1809

Genome size 3,387,40 3,386,76 3,387,73 3,386,56 3,186,62 3,187,30 3,188,35
(bp) 5 7 2 8 8 1 1
GC content
(%) 29.9 29.9 29.9 29.9 30.7 30.7 30.7
No. of contigs 91 89 88 91 118 124 124
No. of
subsystem 243 243 243 243 244 244 244
No. of CDS 3068 3068 3067 3062 2941 2935 2952
No. of RNAs 68 69 70 66 67 62 62
119
Antimicrobial resistance genes
prediction
Overview of AMR genes by
1 1
2
QS group 2 QR group
Total 165 genes Total 173 genes
were predicted were predicted
3 3
4
5 4 5
1. Tetracycline 1. Macrolide
2. Macrolide 2. Tetracycline
3. Fluoroquinolne 3. Fluoroquinolne
4. Peptide antibiotic 4. Glycopeptide antibiotic
120
5. Glycopeptide antibiotic 5. Peptide antibiotic
prediction Quinolone resistance (QR)
associated gens
QS group QR group
▪ Total 45 QR associated genes

were predicted
▪ 40 genes were overlapped
between QS and QR group
3 4 2 ▪ 5 genes were different
0
sdiA,
qnrS2,
smeD,
marR mutant
mexF
121
prediction Quinolone resistance (QR)
associated gens
Resistance
Gene family Genes No.
Mechanisms
acrS, adeL, adeN, cmeA, cmeB, cmeC, acrA,
RND antibiotic efflux acrR, marR mutant, evgA, evgS, gadW, ramR
22 Antibiotic efflux pump
pump mutant, mexB, mexF, mexH, mexV, cpxR, sdiA,
smeD, smeE, soxS mutant
MFS antibiotic efflux arlR, arlS, efmA, emrA, emrB, evgA, evgS, lrfA,
13 Antibiotic efflux pump
pump pmrA, qacA, qacB, qepA2, soxS mutant
MATE transporter cdeA, hmrM 2 Antibiotic efflux pump
ABC antibiotic efflux
efrA, efrB, patB, soxS mutant 4 Antibiotic efflux pump
pump
QR genes gyrB, parC and parE conferring FQR 3 Drug targets alteration
Quinolone resistance
QnrS4, QnrS7, QnrVC7 3 Drug target protection
proteins (Qnr)
* RND: resistance-nodulation-cell division, MFS: major facilitator superfamily,

ABC: ATP-binding cassette, MATE: multidrug and toxic compound extrusion 122
prediction
Quinolone resistance (QR)
associated gens
▪ Total 45 QR associated genes were downloaded from
CARD and blasted against F. columnare genomes by
Blast2GO software
▪ Heat map of similarity percentage were generated
▪ The genes with >50% identity were considered as QR
associated genes
parC conferring FQR

gyrB conferring FQR
E. coli acrR mutant
P. aeruginosa CpxR
parE confering FQR

E. cloacae acrA
marR mutant
ramR mutant
sox S mutant
E. coli acrA
QnrVC7
QepA2
mex H
sme D
hmrM
QnrS2
QnrS4
cme C
cme A
mex V
cme B
mex B
mex F
sme E
gadW
efm A
emrA
pmrA
ade N
emrB
ade L
cde A
qac A
qac B
evg A
pat B
evg S
acrS
sdi A
efrA
arlR
efrB
arlS
CRP
IrfA
CC1802 47 47 72 58 50 48 44 44 43 49 43 50 52 46 58 56 45 50 56 55 49 52 49 46 49 45 42 45 55 56 59 76 49 50 49 45 41 39 49 50 44 51 55
CC1803 47 47 72 58 50 48 44 44 43 49 43 50 52 46 58 56 45 50 56 55 49 52 49 46 49 45 42 45 55 56 59 76 49 50 49 45 41 39 49 50 44 51 55
CC1805 47 47 72 58 50 48 44 44 43 49 43 50 52 46 58 56 45 50 56 55 49 52 49 46 49 45 42 45 55 56 59 76 49 50 49 45 41 39 49 50 44 51 55
CC1808 47 47 72 58 50 48 44 44 43 49 43 50 52 46 58 56 45 50 56 55 49 52 49 46 49 45 42 45 55 56 59 76 49 50 49 45 41 39 49 50 44 51 55
SP1802 49 46 67 54 54 48 68 44 45 48 44 49 56 47 58 58 44 50 55 55 49 71 50 46 51 56 43 45 51 59 59 79 48 50 48 45 37 41 40 53 51 55
SP1805 49 46 67 54 54 48 68 44 45 48 44 49 56 47 58 58 44 50 55 55 49 71 50 46 51 56 43 45 51 59 59 79 48 50 48 45 37 41 40 53 51 55
SP1809 49 46 67 54 54 48 68 44 45 48 44 49 56 47 58 58 44 50 55 55 49 71 50 46 51 56 43 45 51 59 59 79 48 50 48 45 37 41 40 53 51 55
0 50 100
123
Color key
Heat map with similarity percentage of all QR genes
prediction
Quinolone resistance (QR) gens
Gene family Genes

QR group
adeN, cmeA, acrR mutant, marR
22 RND antibiotic
mutant, evgA, evgS, ramR mutant,
efflux pump
mexB, cpxR, smeE, soxS mutant
MFS antibiotic arlR, arlS, emrB, evgA, evgS, lrfA, pmrA,

efflux pump qacA, soxS mutant
QS group MATE transporter hmrM
16 ABC antibiotic
efrA, efrB, patB, soxS mutant
efflux pump
QR genes gyrB, parC and parE conferring FQR
124
Chromosomal mutations
prediction
▪ 13 chromosomal
mutations were
predicted from Resfinder
Resfinder
- 16S_rrsB
- 16S_rrsC
- 16S_rrsH
- 23S
- ampC
- folP
- gyrA
- gyrB
- parC
Mutation identification by - parE
Mega7 software - pmrA
- pmrB
- rpoB
125
Overview of Quinolone resistance mechanism of F. columnare
Quinolone
Quinolone
126
CONCLUSION
▪ Most of F. columnare isolates were resistant to first

generation of quinolone (oxolinic acid and nalidixic
acid)
▪ The main quinolone resistance mechanism of
F. columnare are target-site genes mutation followed by
efflux pump activity
▪ Major type of efflux pump associated with QR is
belonged to RND efflux pump family
▪ Novel mutation in gyrB (Ser370→Asn) and parC
(Ala→183Pro) were discovered in this study
▪ Further research of proteomic are needed
127
Output of this study
128
Research team
Dr. Channarong Dr.

Rodkhum Dr. Ha Thanh
Pattanapon
Dong
Kayansamruaj
Dr. Saengchan Dr. Nopadon Dr. Benjamin

129 Senapin Pirarat LaFrentz
130
131
THANK YOU
FOR YOUR ATTENTION
132

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The Molecular Research in Thailand's Aquaculture

: Pathogen Characterization and

• Appropriate stock density

• Appropriate nutrition for each breed and

• Water qualtity management and

• Supplement with immunostimulants such as

• Use Probiotics or Synbiotics

• Vaccination for important diseases

• Haemorrhagic septicemia such as Aermonas septicemia

• Tilapia Tilapinevirus infection

• Parasitic infection such as Trichodina and Dactylogyrus

• Mixed infection or concurrent infections

Major diseases affecting tilapia

9 Source : http://www.thefishsite.com/articles/139/ diseases-of-tilapia-an-introduction

Diseased fish usually infected by multiple organisms called

Hatchery Nursery Pre-grow out Grow out

0 gram 1 gram 15 gram 100 gram 1.5 Kg

Complied from : https://thefishsite.com/ articles/ diseases-of-farmed-barramundi-in-asia

Disease investigation Molecular techniques and

Isolation and Identification

Assoc. Prof. Dr. Channarong Rodkhum

 Most disease laboratory studies are typically aimed at single

 Occur annually in floating

 Specific PCR diagnosis for internal organs of diseased fish:

Farm 1 (%) 100 100 0 0 0 40 40

1 10 A. veronii NK01 I.P. injection 1.36 × 107 CFUs fish-1

2 10 A. veronii NK06 I.P. injection 0.82 × 107 CFUs fish-1

3 10 S. agalactiae NK13 I.P. injection 2.30 × 108 CFUs fish-1

4 10 P. shigeloides NK11 I.P. injection 1 × 107 CFUs fish-1

5 10 F. columnare NK-Fc01 I.M. injection 1 × 107 CFUs fish-1

6 10 TSB (Control) I.P. injection 0.1 mL sterileTSB

Red tilapia fingerlings model

▪ Francisella noatunensis subsp. orientalis +

Isolation and identification isolates of Flavobacterium

Phenotypic classification Genotypic classification

Summary of the characteristics of Flavobacterium columnare isolated

Geographical locations for sample collection

➢ 16S-RFLP (n=49) (Darwish et al., 2005; Lafrentz et al., 2014)

➢ ISR Sequencing & Phylogenetic analysis (n=25) (Zavaleta et al., 1999)

Genomovar assignment (16S-RFLP)

Genomovar II: 48 isolates

Thai isolates formed 3 clusters 81 F. columnare EK 28

ISR-2 (587-651 bp)- Branch I (n=9) 99 F. columnare LV-339-01

99 F. columnare LV-339-01 16S rDNA sequencing

• CUVET seq (n=19) F. columnare CUVET1241

49 0.03 0.02 0.01 0.00

Good agreement between ISR and 16S rDNA phylogenetic

ISR phylogenetic 16S rDNA phylogenetic

Unique cluster (ISR-G) Unique sub-cluster (IIB)

16S-RFLP result is not correlated to 16S-rDNA phylogenetic

Virulence assay Adherence and persistence assay

Red tilapia fry (~0.39 g)

The results suggested that an inability of the non-rhizoid

• Causing acute mortality in variety of

– Promote bacterial growth and expression of virulence

Acuumulated percent mortality

control 28 ⁰C control 35 ⁰C control 28 ⁰C control 35 ⁰C

Number of viable bacteria resided in brain Accumulated mortality of infected tilapia

High genetic variation among S.

• Streptococcal bacteria are susceptible

• At least 3 species of Streptococcosis associated

• Ricketsia-like organism (Chen et al. 1994)

• Piscirickettsiosis-like syndrome (Mauel & Miller 2002)