- 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
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].
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.
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.
How to cite: Dong HT, Nguyen VV, Mata W, Kayansamruaj P, Senapin S, Nilubol D, Rodkhum C (2016) Diversity of Non-Flavobacterium columnare Bacteria Associated with Columnaris-like Diseased Fish. Thai J Vet Med. 2016. 46(2): 251-259.
Numerous yellow and pink pigmented bacterial isolates had been recovered from diseased fish exhibiting columnaris-like disease in tilapia (Oreochromis spp.) farms in Thailand. The predominant species had been identified as Flavobacterium columnare while taxonomic classification of the remaining isolates and their pathogenic potential remain undetermined. An additional yellow bacterial strain had also been obtained from a koi carp sample showing columnaris-like symptoms. To continue our previous work, this study described the identification of ten representatives of unknown culturable non-Flavobacterium columnare bacteria based on a combination of phenotypic characteristics and nucleotides homology of 16S rRNA gene and subsequently investigated their pathogenicity in Nile tilapia (Oreochromis niloticus) fingerlings. The majority of the yellow pigmented bacteria were identified as Chryseobacterium spp. while the remainders were identified as [Flexibacter] aurantiacus subsp. excathedrus and Flavobacterium indicum. The pink pigmented bacteria were identified as Flectobacillus roseus. Five representative species of the identified bacteria isolated from diseased tilapia were individually subjected to a pathogenicity test in healthy Nile tilapia fingerlings. The experimental challenge results within 14 days revealed that the tested bacteria exhibited low or no virulence to the fish (0-20% cumulative mortality). This suggests that the identified bacteria merely served as opportunistic pathogens that may require stressors for disease manifestation.
How to cite: Peepim, T., Dong, H.T., Senapin, S., Khunraea, P., Rattanarojpong, T (2016) Epr3 is a conserved immunogenic protein among Aeromonas species and able to induce antibody response in Nile tilapia. Aquaculture. 464: 399-409. doi:10.1016/j.aquaculture.2016.07.022
- Aeromonas associated with previous disease outbreaks were retrospectively identified
- Epr3 is a conserved antigen among isolates of A. caviae, A. veronii and A. jandaei
- Epr3 was expressed in both soluble (β-sheet) and insoluble forms (α-helix)
- Soluble form induced higher antibody response in fish compared to insoluble form
- A combination of two forms provoked a strongest antibody response
- Serum from fish immunized with recombinant Epr3 reacted with A. caviae, A. veronii and A. jandaei
The present study retrospectively identified sixteen putative aeromonad isolates associated with previous disease outbreaks in farmed tilapia, striped catfish and an specified fish species. Based on a combination of biochemical characterization and homology identity of 16S rDNA, the majority of the isolates were identified as Aeromonas caviae (n = 10) while the remaining isolates were Aeromonas veronii (n = 4), Aeromonas jandaei (n = 1) and Plesiomonas shigelloides (n = 1). Potential antigenic gene of epr3 from A. caviae isolates was identified and recombinant proteins were expressed in E. coli system for investigation of immunogenicity in vivo. The result indicated the β-sheet domain (comprising amino acid residue 23–171) expressed in a soluble form while the α-helix domain (residues 172–346) of Epr3 protein expressed in an insoluble form. Interestingly, all 15 identified Aeromonas strains naturally produced Epr3 evidenced by Western blot analysis detected with Epr3-specific polyclonal antibodies. Serum collected from the surviving fish after exposure to A. caviae AH15 was able to react with two domains of the recombinant Epr3. Two immunogenic domains of Epr3 had the capability of enhancing fish humoral immunity but a soluble form (β-sheet) induced a higher antibody response compared to an insoluble form (α-helix). A combination of two forms provoked the strongest antibody response as indicated by dot blot assay. Furthermore, Western blot results showed that sera from fish immunized with Epr3 β-sheet domain, α-helix domain, and pool of both domains could also react with specific protein bands from bacterial lysates of A. caviae, A. veronii and A. jandaei. The result suggests that the common immunogenic protein Epr3 is a promising candidate antigen for development of a cross-protective vaccine against different isolates of Aeromonas spp.