Emergence of tilapia lake virus in Thailand and an alternative semi-nested RT-PCR for detection


  • Outbreaks of TiLV associated with massive mortalities in farmed tilapia in Thailand.
  • An alternative semi-nested RT-PCR has been developed for disease diagnosis.
  • In situ hybridization assay revealed multiple tissues tropism of the virus.
  • Transmission electron microscopy revealed intracytoplasmic viral particles.
  • Partial genome of TiLV from Thailand exhibited genetic variations.



The present study reports outbreaks of tilapia lake virus (TiLV), an emerging pathogen causing syncytial hepatitis of tilapia (SHT), in farmed tilapia in Thailand. Occurrence of the virus was confirmed by RT-PCR and nucleotide sequence homology to the TiLV from Israel. Diseased fish exhibited typical histopathological features of syncytial giant cells in the liver examined through H&E and semi-thin sections. Presence of intracytoplasmic viral particles was revealed by TEM. In situ hybridization using a specific DIG-labeled probe derived from a partial genome segment 3 of TiLV genome revealed multiple tissues tropism of the virus including liver, kidney, brain, spleen, gills and connective tissue of muscle. An alternative semi-nested RT-PCR protocol has been developed in this study for disease diagnosis. Additionally, comparative genetic analysis revealed genetic variations of Thailand-originated virus to the Israel TiLV strains, sharing 96.28 to 97.52% nucleotide identity and 97.35 to 98.84% amino acid identity. Outbreaks of TiLV in different continents might signal a serious threat to tilapia aquaculture globally.

Source: http://www.sciencedirect.com/science/article/pii/S0044848617303484


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Aeromonas jandaei and Aeromonas veronii caused disease and mortality in Nile tilapia, Oreochromis niloticus (L.)

Dong et al. 2017 – Journal of Fish Diseases. doi:10.1111/jfd.12617



Diseases caused by motile aeromonads in freshwater fish have been generally assumed to be linked with mainly Aeromonas hydrophila while other species were probably overlooked. Here, we identified two isolates of non-A. hydrophila recovered from Nile tilapia exhibiting disease and mortality after exposed to transport-induced stress and subsequently confirmed their virulence in artificial infection. The bacterial isolates were identified as Aeromonas jandaei and Aeromonas veronii based on phenotypic features and homology of 16S rDNA. Experimental infection revealed that the high dose of A. jandaei (3.7 × 106 CFU fish−1) and A. veronii (8.9 × 106 CFU fish−1) killed 100% of experimental fish within 24 h, while a 10-fold reduction dose killed 70% and 50% of fish, respectively. When the challenge dose was reduced 100-fold, mortality of the fish exposed to A. jandaei and A. veronii decreased to 20% and 10%, respectively. The survivors from the latter dose administration were rechallenged with respective bacterial species. Lower mortality of rechallenged fish (0%–12.5%) compared to the control groups receiving a primary infection (37.5%) suggested that the survivors after primary infection were able to resist secondary infection. Fish exposed to either A. jandaei or A. veronii exhibited similar clinical signs and histological manifestation.

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Recovery of Vibrio harveyi from scale drop and muscle necrosis disease in farmed barramundi, Lates calcarifer in Vietnam


  • Emergence of scale drop and muscle necrosis disease in farmed barramundi in Vietnam
  • Coinfections of culturable and unculturable bacteria were uncovered from diseased fish.
  • A pathogenic V. harveyi strain was identified as the main causative agent.
  • Fish infected with V. harveyi exhibited similar clinical signs and unique histological changes of naturally diseased fish.
  • The role of unculturable bacteria needs further investigation.



Symptoms of scale drop and muscle necrosis have been considered as an emerging problem in farmed barramundi (Lates calcarifer) in Vietnam since 2013. Naturally diseased fish exhibited remarkable external clinical signs of scale loss, muscle degradation and eventually died. The objective of this study was to determine the infectious causative agent of the clinically diseased fish collected from barramundi caged culture in central Vietnam in 2015. Histological examination from naturally sick fish revealed signs of severe necrotic muscles with infiltration of massive immune-related cells, severe hemorrhage and blood congestion in the brain, collapsed kidney tubules and epithelial cells sloughing into the lumen. Five different bacterial species were recovered from diseased fish and putatively identified as Vibrio harveyi, Vibrio tubiashii, Tenacibaculum litopenaei, Tenacibaculum sp. and Cytophaga sp. based on homology of 16S rDNA sequences and biochemical characteristics. Experimental infection revealed that only V. harveyi killed the fish with similar clinical signs and histological changes compared to naturally diseased fish. Additionally, several unculturable bacteria including T. maritimum were also uncovered from DNA extracted from necrotic muscles by species-specific PCR and 16S rDNA clone library sequencing, but their roles in disease manifestation need further investigation.


Barramundi; Scale drop; Muscle necrosis; Vibrio harveyi; Unculturable bacteria

Source: http://www.sciencedirect.com/science/article/pii/S0044848616309000

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Outbreaks of ulcerative disease associated with ranavirus infection in barcoo grunter, Scortum barcoo (McCulloch & Waite)

How to cite: Kayansamruaj, P., Rangsichol, A., Dong, H. T., Rodkhum, C., Maita, M., Katagiri, T. and Pirarat, N. (2017), Outbreaks of ulcerative disease associated with ranavirus infection in barcoo grunter, Scortum barcoo (McCulloch & Waite). J Fish Dis. doi:10.1111/jfd.12606


In 2013, an outbreak of ulcerative disease associated with ranavirus infection occurred in barcoo grunter, Scortum barcoo (McCulloch & Waite), farms in Thailand. Affected fish exhibited extensive haemorrhage and ulceration on skin and muscle. Microscopically, the widespread haemorrhagic ulceration and necrosis were noted in gill, spleen and kidney with the presence of intracytoplasmic eosinophilic inclusion bodies. When healthy barcoo grunter were experimentally challenged via intraperitoneal and oral modes with homogenized tissue of naturally infected fish, gross and microscopic lesions occurred with a cumulative mortality of 70–90%. Both naturally and experimentally infected fish yielded positive results to the ranavirus-specific PCR. The full-length nucleotide sequences of major capsid protein gene of ranaviral isolates were similar to largemouth bass virus (LMBV) and identical to largemouth bass ulcerative syndrome virus (LBUSV), previously reported in farmed largemouth bass (Micropterus salmoides L.), which also produced lethal ulcerative skin lesions. To the best of our knowledge, this is the first report of a LMBV-like infection associated with skin lesions in barcoo grunter, adding to the known examples of ranavirus infection associated with skin ulceration in fish.

Source: http://onlinelibrary.wiley.com/doi/10.1111/jfd.12606/full

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Severe clinical signs of diseased tilapia infected by Streptococcus agalactiae


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Optimized reverse primer for 16S-RFLP analysis and genomovar assignment of Flavobacterium columnare

How to cite: LaFrentz BR, García JC, Dong HT, Waldbieser GC, Rodkhum C, Wong FS, Chang SF (2016) Optimized reverse primer for 16S-RFLP analysis and genomovar assignment of Flavobacterium columnare. Journal of Fish Diseases. [In press].

Interpretive Summary

Flavobacterium columnare is the causative agent of columnaris disease which severely impacts channel catfish production in the USA and has emerged as an important pathogen in the US rainbow trout industry. Our laboratory previously standardized a genetic typing system for F. columnare in which a portion of the 16S rRNA gene is amplified by PCR using specific primers and then digested with an enzyme that will cut the DNA at specific sites. Based on the number and size of DNA fragments generated, isolates of the bacterium are assigned to a genomovar (i.e., genetic type). During routine use of the typing system, it was observed that the 16S rRNA gene of some isolates did not amplify by PCR and could not be assigned to a genomovar. It was hypothesized that there was nucleotide differences in these isolates at the primer binding sites that accounted for the lack of amplification. To test this hypothesis, the 16S rRNA gene from these isolates were sequenced. Each sequence (n = 8) was deposited in GenBank, and the results demonstrated that these isolates exhibited nucleotide differences at the reverse primer binding site. A new reverse primer was designed, tested, and validated for the ability to amplify the 16S rRNA gene from these isolates as well as others. The new primer can be used in place of the original and should allow for genomovar assignment of all F. columnare isolates.


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Efficacy of α-enolase-based DNA vaccine against pathogenic Streptococcus iniae in Nile tilapia (Oreochromis niloticus)

How to cite: Kayansamruaj P, Dong HT, Pirarat N, Nilubol D, Rodkhum C Efficacy of α-enolase-based DNA vaccine against pathogenic Streptococcus iniae in Nile tilapia (Oreochromis niloticus). Aquaculture. http://dx.doi.org/10.1016/j.aquaculture.2016.10.001


  • DNA vaccines (pEno) targeting α-enolase of Streptococcus iniae were constructed.
  • Immunization with pEno can induce fair protection against S. iniae in Nile tilapia.
  • Relative percent survival of vaccinated fish was 63%.
  • Protection was positively related to immune genes expression and serum antibody.


Streptococcus iniae (SI) is an important pathogenic bacterium causing severe mortality in farmed fish worldwide. In the current study, the α-enolase-based DNA vaccine was constructed and examined for its effectiveness against SI infection in Nile tilapia (Oreochromis niloticus). The juvenile tilapia were immunized intramuscularly with DNA vaccine, pEno, and kept for 30 days prior to the intraperitoneal challenge with 2.7 × 107 CFU of pathogenic SI. At two weeks post challenge, the pEno group yielded the highest survival rate at 72.5%, whereas mock vaccination and negative control groups gained only 40 and 25%, respectively. The protection of vaccine tended to be related to the expression of immune-relevant genes (IL-1β, TNF-α, COX-2, IL-12β and IL-13Rα1) at 7 day post-vaccination (dpv) and the anti-SI serum antibody level at 30 dpv (before in vivo challenge). This study indicated that pEno was able to elicit immune responses and conferred protection against streptococcosis associated with SI infection in Nile tilapia.


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