Genome characterization of piscine ‘Scale drop and Muscle Necrosis syndrome’-associated strain of Vibrio harveyi focusing on bacterial virulence determinants

How to cite

Kayansamruaj, P., Dong, H. T., Hirono, I., Kondo, H., Senapin, S. and Rodkhum, C. (2018), Genome characterization of piscine ‘Scale drop and Muscle Necrosis syndrome’-associated strain of Vibrio harveyi focusing on bacterial virulence determinants. J Appl Microbiol. doi:10.1111/jam.13676



Genomic characterization of Harveyi clade vibrio strain Y6 causing ‘Scale drop and Muscle Necrosis syndrome’ (SDMN) isolated from barramundi (Lates calcarifer) in Vietnam.

Methods and Results

A bacterial genome was sequenced using Illumina MiSeq platform. Multilocus sequence analysis confirmed that the bacterium belongs to Vibrio harveyi species. Further phylogenetic analysis inferred from core genome SNPs revealed a close relationship between our bacterium and the V. harveyi isolated from groupers in Taiwan and China. BLASTp results indicated that V. harveyipiscine strains carried numerous adhesin, secretion system, siderophore and toxin-related genes. Genome comparison between Y6 and thirty-two strains of V. harveyi from different origins showed that at least 17 potential virulence genes were present exclusively in the strain Y6. Many of these (6 out of 17 genes) were homologous to pyoverdine siderophore, a secreted high-affinity iron chelator, clusters originally found in Pseudomonas aeruginosa. Genome of V. harveyi Y6 was incorporated by a bacteriophage VHY6φ and replication protein of the phage was most similar to CTXφ described previously in V. cholera and V. fischeri. However, the cholera toxin-encoding genes, namely ctxA and ctxB, were absent from VHY6φ, while the CTXφ-enterotoxin gene (zonula occluden toxin; zot) remained intact.


Several putative virulence genes and a phage carrying toxin gene were identified in the genomes of SDMN-associated V. harveyi Y6.

Significance and Impact of the Study

This study confers genomic information of the piscine pathogenic V. harveyi which recently caused widespread mortality. Such information is of importance to gain insight into bacterial molecular pathogenesis.



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Inapparent infection cases of tilapia lake virus (TiLV) in farmed tilapia


  • Detection of TiLV in clinically healthy adult and fingerling tilapia
  • Histopathology resembling SHT was observed from TiLV-infected fish
  • Low viral load in fish tissues was determined by RT-PCR
  • Investigation of inapparent infection should be included in TiLV surveillance program


Tilapia farming has been affected by a newly discovered Orthomyxovirus-like, tilapia lake virus (TiLV), which has caused considerable economic loss to farmers. Currently, mortality-associated TiLV infections have been reported in tilapia farms in Israel, Ecuador, Colombia, Egypt, Thailand, Chinese Taipei, India and Malaysia. In this study, sets of samples collected from clinically healthy adult and fingerling tilapia with no signs of diseases or mortality were randomly diagnosed for TiLV. The tissue samples were examined by semi-nested RT-PCR, histopathology, and in situ hybridization (ISH). Unexpectedly, individual organs (liver, kidney, spleen, brain, and heart) of the tested adult fish (2/2) and liver of the fingerlings (9/19) exhibited positive results in the second step RT-PCR, indicating a low viral load of TiLV in the fish tissues. Sequencing analysis of 250-bp amplicons revealed 97.2% identity to the prototype strain from Israel. Histopathology was investigated in the adult fish specimens and pathological features resembling syncytial hepatitis were observed while ISH yielded no detectable signal. Unlike previous reports, this study revealed cases of inapparent or subclinical infections of TiLV in tilapia. Underlying factors and mechanisms between host and virus resulting in inapparent infection require further scientific investigation.


Keywords: Inapparent infection; TiLV; Tilapia lake virus; RT-PCR



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Piper betle Leaf Extract Inhibits Multiple Aquatic Bacterial Pathogens and In Vivo Streptococcus agalactiae Infection in Nile tilapia


An in vitro assessment of antimicrobial properties of aqueous and ethanol extracts from solo garlic (Allium sativum), garlic chive (Allium tuberosum) and betel leaves (Piper betle) on six bacterial pathogens in aquaculture, and a challenge of Nile tilapia, Oreochromis niloticus with Streptococcus agalactiae were performed. Generally, minimum inhibitory concentrations (MIC) ranged from 26.63 to 53.25 mg mL-1 for aqueous solo garlic (G) and 14.60 to 29.20 mg mL-1 for garlic chive extracts for all pathogens tested. Ethanol extract of betel leaves (P) exhibited the strongest antibacterial activity (0.15 – 0.60 mg mL- 1). P and G incorporated in feed at high and low doses as multiples of MIC [High; H (10X for PH and 3X for GH) and Low; L (3X for PL and 1X for GL)] were fed to tilapia followed by in vivo challenge against S. agalactiae (1 x 108 CFU mL-1). Ethanol extract of P. betle significantly improved survival (P < 0.05; PH=100%, PL =77%). White blood cells (WBC), lymphocytes and monocytes differed significantly (P < 0.05) among treatments and the highest WBC value (1.175 × 103) was for PH. Use of ethanol extract of Piper betle seems promising for sustainable disease management in aquaculture.

Keywords: herbal, antimicrobial, risk, haematology, survival

Source: Turkish Journal of Fisheries and Aquatic Sciences. DOI: 10.4194/1303-2712-v18_5_03

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Evidence of TiLV infection in tilapia hatcheries from 2012 to 2017 reveals probable global spread of the disease

TiLV-Histopathology HT Dong

Histopathological feature of syncytial hepatitis of tilapia (SHT) observed from the liver of  TiLV-infected tilapia (Photo: HT Dong)


Recent outbreaks of tilapia lake virus (TiLV) in farmed tilapia in Thailand were the first indication of spread of the virus to the Southeast Asia region. Here we further investigate TiLV infection of archived and newly collected fish samples obtained from Thai hatcheries from 2012 to 2017. Fertilized eggs, yolk-sac larvae, fries, and fingerlings
were tested for the TiLV using an established semi-nested RT-PCR assay. The results revealed that the majority of the tested samples were TiLV positive, including our earliest preserved samples collected in year 2012. DNA sequence analysis of representative amplified products also confirmed the presence of TiLV. Since the
discovery of TiLV in 2012, over 40 countries worldwide have imported tilapia fry and fingerlings, and some may have been unaware of risk that they might be infected with TiLV. Thus, if they have not already done so, we recommend that countries that have imported tilapia for aquaculture carry out surveillance studies for its presence and also add TiLV to their import quarantine inspection list.

Keywords: Disease transmission, TiLV, Tilapia hatcheries


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Infectious spleen and kidney necrosis disease (ISKND) outbreaks in farmed barramundi (Lates calcarifer) in Vietnam


  • The first report on emergence of ISKD in farmed barramundi from Vietnam.
  • The causative agent was identified as Megalocytivirus ISKNV genotype II.
  • A Megalocytivirus RSIV vaccine conferred only partial protection.
  • Structural analysis revealed significant differences between the two viruses.


Emergence of a disease with clinical signs resembling megalocytivirus infection seriously affected large-scale barramundi farms in Vietnam in 2012–2014 with estimated losses reaching $435,810 per year. An oil-based, inactivated vaccine against red sea bream iridovirus (RSIV) was applied in one farm for disease prevention without analysis of the causative agent, and the farmer reported inadequate protection. Here we describe histological and molecular analysis of the diseased fish. PCR targeting the major capsid protein (MCP) of megalocytiviruses yielded an amplicon with high sequence identity to infectious spleen and kidney necrosis virus (ISKNV) genotype II previously reported from other marine fish but not barramundi. Detection of the virus was confirmed by positive in situ hybridization results with fish tissue lesions of the kidney, liver, pancreas, and brain of the PCR-positive samples. Based on the complete sequence of the MCP gene, the isolate showed 95.2% nucleotide sequence identity and 98.7% amino acid sequence identity (6 residue differences) with the MCP of RSIV. Prediction of antigenic determinants for MCP antigens indicated that the 6 residue differences would result in a significant difference in antigenicity of the two proteins. This was confirmed by automated homology modeling in which structure superimpositioning revealed several unique epitopes in the barramundi isolate. This probably accounted for the low efficiency of the RSIV vaccine when tested by the farmer.


Barramundi; ISKNV; Lates calcarifer; RSIV; Vaccine


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Comparative genome analysis of fish pathogen Flavobacterium columnare reveals extensive sequence diversity within the species

How to cite: Pattanapon Kayansamruaj, Ha Thanh Dong, Ikuo Hirono,
Hidehiro Kondo, Saengchan Senapin, Channarong Rodkhum , Comparative genome
analysis of fish pathogen Flavobacterium columnare reveals extensive sequence diversity within the species. Infection, Genetics and Evolution (2017), doi: 10.1016/


  • Comparative genomics among fish pathogenic F. columnare strains was investigated
  • Virulence genes were equally distributed among F. columnare strains.
  • Phylogenetic analyses indicated extensive genetic diversity within the species.
  • 16S–RFLP failed to distinguish between genomovar II isolate groups.
  • Tilapia-originated strains from Thailand would be distinct taxonomic groups.



Flavobacterium columnare is one of the deadliest fish pathogens causing devastating mortality in various freshwater fish species globally. To gain an insight into bacterial genomic contents and structures, comparative genome analyses were performed using the reference and newly sequenced genomes of F. columnare including genomovar I, II and I/II strains isolated from Thailand, Europe and the USA. Bacterial genomes varied in size from 3.09 to 3.39 Mb (2714 to 3101 CDSs). The pan-genome analysis revealed open pan-genome nature of F. columnare strains, which possessed at least 4953 genes and tended to increase progressively with the addition of a new genome. Genomic islands (GIs) present in bacterial genomes were diverse, in which 65% (39 out of 60) of possible GIs were strain-specific. A CRISPR/cas investigation indicated at least two different CRISPR systems with varied spacer profiles. On the other hand, putative virulence genes, including those related to gliding motility, type IX secretion system (T9SS), outer membrane proteins (Omp), were equally distributed among F. columnare strains. The MLSA scheme categorized bacterial strains into nine different sequence types (ST 9–17). Phylogenetic analyses based on either 16S rRNA, MLSA and concatenated SNPs of core genome revealed the diversity of F. columnare strains. DNA homology analysis indicated that the estimated digital DNA-DNA hybridization (dDDH) between strains of genomovar I and II can be as low as 42.6%, while the three uniquely tilapia-originated strains from Thailand (1214, NK01 and 1215) were clearly dissimilar to other F. columnare strains as the dDDH values were only 27.7–30.4%. Collectively, this extensive diversity among bacterial strains suggested that species designation of F. columnare would potentially require re-emendation.


Comparative genomics; Flavobacterium Columnare; Genetic diversity; Pan-genome; Taxonomy


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A natural Vibrio parahaemolyticus ΔpirAVp pirBVp+ mutant kills shrimp but produces neither PirVp toxins nor acute hepatopancreatic necrosis disease lesions

How to cite: Phiwsaiya K, Charoensapsri W, Taengphu S, Dong HT, Sangsuriya P, Nguyen GTT, Pham HQ, Amparyup P, Sritunyalucksana K, Taengchaiyaphum S, Chaivisuthangkura P, Longyant S, Sithigorngul P, Senapin S. 2017. A natural Vibrio parahaemolyticus ΔpirAVp pirBVp+ mutant kills shrimp but produces neither PirVp toxins nor acute hepatopancreatic necrosis disease lesions. Appl Environ Microbiol 83:e00680-17.


Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor a pVA plasmid encoding toxins PirAVp and PirBVp. These are released from VPAHPND isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that V. parahaemolyticus isolate XN87 lacked pirAVp but carried pirBVp. Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirBVp was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VPAHPND isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirAVp gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirAVp gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirAVp gene sequence and in the downstream pirBVp gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirABVp mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However,
the virulence of XN87 revealed that some V. parahaemolyticus isolates carrying mutant pVA plasmids that produce no PirVp toxins can cause mortality in shrimp in
ponds experiencing an outbreak of early mortality syndrome (EMS) but may not
have been previously recognized to be AHPND related because they did not cause
pathognomonic AHPND lesions.


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