Histopathology and culturable bacteria associated with “big belly” and “skin nodule” syndromes in ornamental Siamese fighting fish, Betta splendens

How to cite: Dong HT, Senapin S, Phiwsaiya K, Techatanakitarnan C, Dokladda K, Ruenwongsa P, Panijpan B (2018) Histopathology and culturable bacteria associated with “big belly” and “skin nodule” syndromes in ornamental Siamese fighting fish, Betta splendens. Microbial Pathogenesis. DOI: 10.1016/j.micpath.2018.06.005.



The Siamese fighting fish (Betta splendens) is one of the popular aquarium ornamental fish in the global trade. Large numbers of ornamental fish farmed in central Thailand suffered from two common syndromes; preliminarily named skin nodule syndrome (SNS) and big belly syndrome (BBS): they showed noticeable clinical signs of abnormal appearances resulting in depressed saleability. Since very few specifics are known about causative agents of these syndromes, this study aimed at investigating histopathological features and culturable bacteria associated with these fish infected in the process of farming. Histopathologically, SNS fish consistently exhibited necrosis and severe melanization in the muscles and multiple internal organs. Whereas BBS fish exhibited either typical granulomas or tissue damage associated with acid-fast stained bacteria and Gram negative bacteria, respectively. Six different Gram negative bacterial species were recovered from BBS fish while 23 bacterial species belonging to 14 genera were recovered from fish suffering from SNS. Most of the culturable bacteria are new to betta fish and some of them are known to be marine bacteria, suggesting possible entry route via a contaminated live feed, commercial Artemia shrimp. The true causative agents of these syndromes remain unclear. However, histopathological changes and existence of a wide range of bacteria associated with the naturally diseased fish suggest involvement of multiple bacterial infections.

Keywords: Betta splendens; Big belly syndrome; Culturable bacteria; Histopathology; Skin nodule syndrome

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Quinolone-resistant phenotype of Flavobacterium columnare isolates harbored point mutations in both parC and gyrA but not in either gyrB or parE

How to cite: Mata W, Putita C, Dong HT, Kayansamruaj P, Senapin S, Rodkhum C (2018) Quinolone-resistant phenotype of Flavobacterium columnare isolates harbored  point mutations in both parC and gyrA but not in either gyrB or parE. Global Antimicrobial Resistance. doi: 10.1016/j.jgar.2018.05.014


Objective: Determination of mutations associated with quinolone-resistant (QR) phenotype of Flavobacterium columnare isolates.

Methods: The susceptibility of F. columnare isolates (n = 53) to 11 antibiotics, including 2 quinolones, was investigated using disk diffusion method. Oxolinic acid was subsequently chosen for minimum inhibitory concentration (MIC) assay. PCR and sequence analysis of four genes involved in the quinolone resistance-determining regions (QRDRs) from OA-resistant F. columnare compared to that of susceptible isolates were subsequently investigated.

Results: The result of disk diffusion assay revealed that the majority of isolates was susceptible to all tested antibiotics. However, 14 and 8 isolates were resistant to 2 quinolone antibiotics; oxolinic acid (OA) and nalidixic acid (NA), respectively. No multiple drug resistance was found in this study. MIC assay revealed 4 additional isolates that were resistant to OA (≥4 μg mL−1), making a total of 18 OA-resistant isolates obtained in this study. The results of DNA sequencing showed that missense mutations in both parC and gyrA but not in either gyrB or parE were identified in QR F. columnare isolates. Mutation in parC resulted in a change in His87-Tyr. For gyrA, 15 isolates of Thai origins exhibited change at residue 83 from Ser to either Phe, Tyr or Ala, whereas 3 Vietnamese isolates contained two mutation sites, Ser83-Ala and Asp87-Tyr.

Conclusion: This study is the first to reveal that QR phenotype of F. columnare isolates harbored missense mutations in both parC and gyrA but not in gyrB and parE of the QRDRs.

Source: https://www.sciencedirect.com/science/article/pii/S2213716518300985

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C-terminal domain of WSSV VP37 is responsible for shrimp haemocytes binding which can be inhibited by sulfated galactan

How to cite: Sotanon N, Saleeart A, Rattanarojpong T, Dong HT, Senapin S, Wongprasert K, Sarikavanij S, Khunrae P (2018) C-terminal domain of WSSV VP37 is responsible for shrimp haemocytes binding which can be inhibited by sulfated galactan. Fish and Shellfish Immunology. 77: 312-318


Viral envelope proteins play an important role in facilitating the attachment of viruses to the surface of host cells. Here, we investigated the binding of White Spot Syndrome Virus (WSSV) VP37 to haemocytes of whiteleg shrimp, Litopenaeus vannamei. Three versions of recombinant VP37 proteins, including full length VP37 (VP37(1-281)), C-terminal domain VP37 (VP37(111-281)) and C-terminal domain disrupted VP37 (VP37(1-250)) were individually expressed and tested for their haemocytes binding ability. Through an ELISA-based binding assay, we found that VP37(111-281) bound to shrimp haemocytes in a similar way to VP37(1-281), while VP37(1-250) exhibited a significantly weaker binding. This suggests that the C-terminal domain of VP37 is required for the binding of VP37 to shrimp haemocytes. Furthermore, we found that the binding of VP37 to shrimp haemocytes was impaired by pre-incubation of VP37 with sulfated galactan (SG), a sulfated polysaccharide derived from red seaweed (Gracilaria fisheri). Previously, it has been shown that a type of sulfated polysaccharide, heparin, is also present in L. vannamei. To investigate the role of heparin as a receptor for VP37, the binding of VP37 to porcine heparin, whose structure is similar to that found in L.vannamei, was investigated in a Surface Plasmon Resonance (SPR) system. The results showed that VP37 bound strongly to heparin with binding affinity (KD) of 1.0 μM and the binding was significantly blocked by SG. These findings have lead us to propose that the attachment of WSSV might be mediated by the interaction between VP37 and a heparin-like molecule presented on the shrimp cells.


Heparin; Shrimp; Sulfated galactan; VP37; WSSV

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Tilapia lake virus: a threat to the global tilapia industry?

How to Cite: Jansen MD, Dong HT, Mohan CV (2018) Tilapia lake virus: a threat to the global tilapia industry? Review in Aquaculture. https://doi.org/10.1111/raq.12254 [open access].


Photomicrographs of haematoxylin and eosin‐stained sections of tissue from the liver, kidney and brain of normal fish (a, e, g) and TiLV‐infected fish (b–d, f, h). The infected liver tissue showed syncytial hepatocytes and foamy cytoplasm (b), intracytoplasmic inclusion bodies (c) and inflammation with pancreatic necrosis (d). Kidney tissue showed syncytial cells and severe infiltration of inflammatory lymphocytes (f). Brain tissue also showed syncytial cells and severe infiltration of inflammatory lymphocytes (h).


Tilapia lake virus (TiLV) is a recently described virus affecting wild and farmed tilapines. At present, it has been reported on three continents (Asia, Africa and South America) and the number of countries where the agent has been detected is likely to increase rapidly as a result of increased awareness, surveillance and availability of diagnostic methods. Any lack of openness regarding the TiLV status of a translocating live tilapia population destined for aquaculture may inadvertently contribute to the spread of the agent. Currently, there is no cure for viral diseases in aquaculture and while vaccines and selective breeding have proved successful in reducing the severity of some viral diseases, there are currently severe knowledge gaps relating to TiLV and no effective, affordable vaccines are yet available. This paper summarizes the published scientific information on TiLV and highlights important issues relating to its diagnosis, mitigation and control measures. While there have been no scientific studies on the socio‐economic impact of TiLV, it may pose a significant threat particularly to small‐scale fish farmers’ livelihoods and wild tilapine populations if left uncontrolled. To aid disease investigations, the authors propose case definitions for suspected and confirmed cases of TiLV infections.

Source: https://onlinelibrary.wiley.com/doi/full/10.1111/raq.12254

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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


Aims: 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.

Conclusions: 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.

Source: http://onlinelibrary.wiley.com/doi/10.1111/jam.13676/abstract


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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

Source: https://www.sciencedirect.com/science/article/pii/S0044848617323761


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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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