là Äoạn gene sá» 3 (segment 3). 3.2. Nguyên liá»u hóa há»c và sinh há»c. 3.2.1. Nguyên liá»u hóa há»c. - Dung dá»ch Phosphate Buffered Saline (PBS):. Thà nh phần.
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Overview of current and emerging infectious diseases in farmed tilapia
Ha Thanh Dong & Saengchan Senapin King Mongkut‟s University of Technology Thonburi Fish Health Platform, CENTEX Shrimp, BIOTEC Mahidol University
29-30 September 2017
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Aquaculture production Aquaculture - the most important food animal-producing sector
FAO, 2014
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Tilapia production
Fitzsimmons, University of Arizona
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Tilapia Aquaculture • Intensive culture forms are highly developed corresponding to the leading position. • In Asia, tilapia is a main aquaculture species
Open cage-cultured system
Pond cultured system
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Dr. Marian McLoughlin (Fish Vet Group)
Disease is considered as one of the major problems to aquaculture production globally
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Current & Emerging Viral Diseases Viral nervous necrosis (VNN) disease Betanodavirus Tilapia larvae encephalitis virus (TLEV) disease TLEV (herpes-like virus) Infectious spleen & kidney necrosis virus disease (ISKND)/ Iridoviral disease ISKNV (belongs to Megalocytivirus) Tilapia lake virus disease (TiLVD)/Syncytial hepatitis of tilapia (SHT)/ Summer mortality syndrome/One month mortality syndrome TiLV=Orthomyxo-like virus
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Viral Nervous Necrosis (VNN) disease • VNN has been reported in more than 30 species both freshwater and marine fish (mainly in marine fish)
• Several cases of infections have been reported in tilapia • In 2011, a case occurred in Thailand ▫ 10 days-old larvae of tilapia ▫ Mortality 90-100% ▫ signs of neurological disorders: loss of balance, erratic swimming ▫ Histopathological manifestation of VNN disease ▫ 93.07–93.88% similarity to red-spotted grouper nervous necrosis virus (RGNNV)
Lack of surveillance in AsiaPacific region
Vacuolation was observed in brain, eye and spinal cord of diseased fish. Immunohistochemistry showed positive nodavirus antigens in the affected neuron
(Keawcharoen et al. JFD 2015, 38, 49-54)
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Tilapia larvae encephalitis virus (TLEV) disease • Causative agent: TLEV/Herpes-like virus • Clinical signs: spiral swimming behaviour • Host: blue tilapia (O. aureus), red tilapia (Oreochromis sp.), Nile tilapia (O. niloticus) • Mortality: variable, reach up to 98% • Susceptible stages: 32-34 days post fertilization • Geographical distribution: Israel • Histopathological feature: Not available • PCR detection: available TLEV-1(5′ TCGTGGGCCTTATCCCGCGT 3‟) TLEV-2 (5′ GAGACCAGAAAGTGCTTCTC 3′)
Lack of investigation in AsiaPacific region! Shlapobersky et al. Virology (2010) 399: 239-247
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Infectious spleen and kidney necrosis disease • Synonym: Iridovirus disease (common name), red sea bream iridoviral disease (OIE) • Causative agent: ISKNV-Megalocytivirus • Clinical signs: darkening, pale gills (not typical) • Host: wide range of both marine and freshwater fish, including tilapia • A case in USA (Subramaniam et al. 2016) ▫ Tilapia fry/fingerlings, Mortality 50-75%
• In Thailand ▫ Multiple infections of ISKNV/Iridovirus was reported in cage culture & a semi-nested PCR was developed (Dong et al. 2016) ▫ Recent reports: vertical transmission & LAMP detection method
• In Vietnam ▫ An outbreak in sea bass (Dong et al. 2017) ▫ No report in tilapia
Subramaniam et al. (2016)
Lack of surveillance & virulence test
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Tilapia lake virus disease (TiLVD) • Causative agent: TiLV (Orthomyxo-like virus) • Clinical signs: variable, nonspecific • Host: blue tilapia (O. aureus), red tilapia (Oreochromis sp.), Nile tilapia (O. niloticus) • Mortality: variable, 6.4-90% • Susceptible stages: 32-34 days post fertilization • Geographical distribution: Israel, Ecuador, Colombia, Egypt, Thailand, Taiwan (plus India, Vietnam, Malaysia, Bangladesh) • Histopathological feature: Syncytial hepatitis, intracytoplasmic inclusion bodies in the liver • Detection methods
Ferguson et al. 2014
▫ RT-PCR, real-time ▫ In situ hybridization ▫ Cell culture
• Vaccine: in progress
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Bacterial Diseases in Farmed Tilapia in Thailand Current Diseases Streptococcosis Streptococcus agalactiae Streptococcus iniae Lactococcus garviae
Emerging Diseases Francisellosis Francisella noatunensis subsp. orientalis
Hahellosis (red egg disease)
Columnaris Flavobacterium columnare Non-F. columnare bacteria
Hahella chejuensis
Motile Aeromonads (Non-A. hydrophila) Aeromonas veronii
Motile Aeromonads Aeromonas hydrophila A. sobria
Aeromonas jandaei
Miscellaneous disease
Edwardsiellosis
F. columnare, A. veronii, S. agalactiae & Megalocytivirus
Edwardsiella ictaluri Edwardsiella tarda
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Columnaris Disease • Causative agent: Flavobacterium columnare • Clinical signs: fin rot, gills necrosis, muscle necrosis • Host: red and Nile tilapia • Mortality: variable, reached up to 100% in challenge experiments Clinical signs
Noted F. columnare is an opportunistic pathogen Disease usually occurs after transportation Cage culture is more vulnerable
Strain caused disease in tilapia should be reclassified as a new species No vaccine available Dong et al. J Fish Dis (2015) 38:901-913
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Columnaris Disease 50
Unique cluster
G
83
Thai isolates (25) Br anch II
F. columnare BZ-04 99
Branc h II F
97 F. columnare BZ-01 99 F. columnare BZ-02
E
F. columnare BZ-05 99 F. columnare CUVET1231 100 F. columnare CUVET1232
D
F. columnare ALG-00-530
96
81 F. columnare EK 28 64 F. columnare LP 8
Phylogenetic tree based on ISR of F. columnare isolates
F. columnare LV-339-01
99
F. columnare CUVET1360 F. columnare CUVET1363 98
C
F. columnare CUVET1365 F. columnare CUVET1368 F. columnare CUVET1372 F. columnare CUVET1371
96 68
nchcIh I BrBaran
100 F. columnare ATCC 49513 F. columnare ATCC 23463 77
B
F. columnare FK 401
99 100
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
F. columnare CUVET1215 Flavobacterium johnsoniae ATCC 17061 Flavobacterium psychrophilum ATCC 49418
0.05
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Columnaris Disease Flavobacterium columnare ATCC 49512
Dominant Clade
9954
Flavobacterium columnare ATCC 23463 clone GI-1 Flavobacterium columnare F10-HK-A clone GI/II-1 Flavobacterium columnare IAM 14301
50 98 79
71 97
Flavobacterium columnare EK28
Flavobacterium columnare LV339-01 Flavobacterium columnare Ga-6-93 60
63 47
Cold water fish
Flavobacterium columnare ATCC 49513
Flavobacterium columnare LP8
93
99
Genomovar I & I/II
Flavobacterium columnare FK 401
Flavobacterium columnare ALG-00-530 clone GII-18 Flavobacterium columnare PT-14-00-151 clone GIIB-1
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Striped Catfish isolates (n=8)
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Tilapia isolates (n=50)
99
Genomovar II & II-B
Warm water fish
Flavobacterium columnare CUVET1215
Phylogenetic tree based on16S rRNA sequences
Flavobacterium columnare AU-98-24
99
Flavobacterium columnare PH-97028
94 97 54
Genomovar III
Flavobacterium columnare GA-02-14 clone GIII-1 Flavobacterium columnare ARS-1 clone GIA-1 Flavobacterium johnsoniae ATCC 23107
Flavobacterium psychrophilum strain ATCC 49418 74
Flavobacterium branchiophilum NBRC 15030.
0.01
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Columnaris Disease • Standard genomovar typing (16S-RFLP) was unable to applied to tilapia strains from Thailand • We collaborated with Dr. LaFrentz (USDA) to re-optimize the method
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Columnaris Disease
In collaboration with a Japanese research group
Kayansamruaj et al. Infection, Genetics and Evolution 54 (2017) 7–17
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Columnaris Disease
In collaboration with a Japanese research group
Digital DNA-DNA hybridization (dDDH) support taxonomic reclassification of Fc originated from tilapia
Kayansamruaj et al. Infection, Genetics and Evolution 54 (2017) 7–17
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There are something strange?
Rhizoid morphotype
Non-Rhizoid morphotype
What is correlation between Morphotypes & Virulence of F. columnare?
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Columnaris Disease
Rhizoid
Non-Rhizoid
Naturally diseased fish
Experimentally diseased fish
Rhizoid morphotype is pathogenic Non-Rz morphotype is non-pathogenic
Adhesion & Persistence Dong et al. J Fish Dis (2016) 39:649-655
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Columnaris Disease Prevention and Treatment
From laboratory to fish farms
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Ignore OR Find Answer?
Who are you?
F. columnare
Columnaris Disease Diversity of Non-F. columnare associated with columnaris diseased fish
Phylogenetic analysis based on 16S rRNA
Most bacteria first found in tilapia Experimental challenge (I.M.) showed 0-20% mortality May serve as opportunistic pathogens Dong et al. Thai J Vet Med (2016) 46:251-259
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…the end is a new beginning
How important of archived samples!
Francisellosis Occurrence of Francisellosis in Thailand
A typical granuloma (H&E)
Intracellular bacteria in macrophages (Giemsa)
First reported occurrence of Francisellosis in Thailand
F. noatunensis subsp. orientalis was confirmed as the causative agent Fulfill Koch‟s postulates
Genus-specific PCR Nguyen et al. Res (2015):1-6
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Francisellosis
Geographical locations of Francisellosis F. noatunensis subsp. noatunensis (cold) F. noatunensis subsp. orientalis (warm) Nguyen et al. Aquac Res (2015):1-6
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Francisellosis
Which one infected with F. noatunensis subsp. orientalis ?
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Francisellosis Whether Francisellosis affect farmed tilapia in Vietnam? Highly predictable A case of Francisellosis was confirmed in our lab PCR diagnosis for species level are available (Dong et al. 2016)
Typical granulomas found in tilapia samples from Vietnam
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The end is a new beginning
FNO is a highly fastidious bacterium A conventional PCR specific for FNO has not yet been established Paraffin section based diagnosis is needed
……go with simple techniques that most laboratories can apply
Duplex PCR assay and in situ hybridization for detection of Francisella spp. and Francisella noatunensis subsp. orientalis in red tilapia
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Francisellosis Development of DNA-based detection method
Design primers & Probe
Specificity of duplex PCR assay Dong et al. Dis Aquat Org (2016) 120: 39-47
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Francisellosis Development of DNA-based detection method
Sensitivity of duplex PCR assay
Tested clinical samples
Dong et al. Dis Aquat Org (2016) 120: 39-47
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Francisellosis Localization of Fno in fish tissue
In situ hybridization using FNO-specific probe revealed positive signals in different organs including the spleen, liver, kidney, gills and intestine Dong et al. Dis Aquat Org (2016) 120: 39-47
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….a negative result story
Francisella noatunensis subsp. orientalis infects striped catfish (Pangasianodon hypophthalmus) and common carp (Cyprinus carpio) but does not kill the hosts
Aquaculture (2016) 464:190–195
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Francisellosis Susceptibility of Striped catfish & Carp to Fno
Francisella-specific PCR assay at day 21 post challenge
In situ hybridization revealed presence of bacteria in day 21 post infection
Striped catfish (ca Tra) and Common carp (ca chep) may served as reservoir Dong et al. Aquaculture (2016) 464:190–195
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Francisellosis Prevention & Treatment Disease outbreaks were associated with cool season
(30 C, no mortality occurred Very chronic, disease outbreaks depend on environmental factors Antibiotics have been using for treatment Commercial vaccine is NOT available
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Hahellosis = Red egg disease
Normal eggs
Red eggs
Occurred in a tilapia hatchery in Thailand since 2010 Mortality 10-50% Normal occur during cold season (
