Assessing the response of Asian seabass (Lates calcarifer) to vaccination against Streptococcus iniae using ELISA

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Vaccination is an important tool to protect farmed animals against contagious diseases. Though common in livestock and domestic animals, vaccination of cultured fish has been more limited1. A recent publication from Tinh et al at the Asian Institute of Technology in Thailand investigated the ability of young Asian seabass (Lates calcarifer, Bloch 1790) to mount an immune response to a vaccine to the common pathogen Streptococcus iniae2. ELISAs performed using the Ao Absorbance Microplate Reader (Catalog # AC3000) from Azure Biosystems were an integral part of the work.

ELISA stands for enzyme-linked immunosorbent assay. The ELISA assay is a research method for quantifying proteins or antigens in an unknown solution, or in medicine as a diagnostic tool.

Learn more about ELISA >

Lates calcarifer and its response to the bacterium Streptococcus iniae

The Asian seabass is a commercially significant species for aquaculture in the coastal waters of Southeast Asia. Like other fish species, this cultured fish species is subject to disease outbreaks from bacterial and viral infections. A significant pathogen of cultured Asian seabass is the bacterium Streptococcus iniae. S. iniae can infect other fish and mammals, such as humans and dolphins, causing substantial mortality rates, skin lesions, and systemic septicemia. To protect the fish (and subsequently human fish farmers) from S. iniae, vaccination is necessary.

Vaccination would ideally take place early in the fish lifespan, before exposure to the pathogen. However, much remains unknown about timing of the development of the adaptive immune system in young Asian seabass and whether young fish can respond to vaccination.B

Studying the immune response to S. iniae

In recent work, Vinh et al investigated whether young Asian seabass could mount an immune response to a vaccine against S. iniae. Fish were vaccinated at 35 or 42 days after hatching (dph) using a heat-killed S. iniae vaccine delivered through immersion immunization (the vaccine was added to the water in which the fish were kept). The immune response of the vaccinated fish was assessed by measuring fish production of IgM by ELISA, and by measuring the expression of several immune-related genes.

The ELISA experiments assessed the amount of antibodies the fish produced that targeted an S. iniae antigen. Plates were coated with antigen and then incubated with antibody extract prepared from the immunized fish at predetermined times after immunization. Six fish were assessed at zero, seven, and 14 days after immunization. Fish antibody was bound to the plate and detected using an anti-Asian seabass primary antibody and a goat-anti-mouse secondary antibody conjugated to HRP. The amount of bound secondary antibody was determined by measuring the absorbance of a chromogenic substrate (3,3′,5,5′-tetramethylbenzidine (TMB)) at 450nm on the Ao Absorbance Microplate Reader.

Azure Biosystems Ao Absorbance Microplate Plate Reader is used for ELISA, Bradford Assays, and more
Trust your data. With the Ao Microplate Reader you will not sacrifice speed for accuracy. With a read speed of <6 seconds, and an accuracy of <0.005 ± 1% (0-3) OD, you will quickly get your results and know they are correct.

The study's findings

The ELISA experiments demonstrated that immunization resulted in a significant increase in production of antibodies targeting S. iniae in the young Asian seabass. Of the fish that were 35 dph, four of six fish generated antibodies to S. iniae antigen 14 days after immunization. Of the fish that were 42 dph, two out of 6 had antibodies to S. iniae antigen seven days after immunization, and three out of six had antibodies 14 days after immunization.

The gene expression experiments similarly demonstrated that expression of relevant genes increased in the young fish.  Expression changes were detected 1 day after immunization in the 42 dph fish compared to seven days in the 35 dph fish. No assays were performed between one and seven days, so any expression changes during that period could not be determined.

The authors conclude that early vaccination of Asian seabass is feasible and 35 dph Asian seabass can acquire immunity against a bacterial pathogen. The immersion immunization approach was successful with these young fish, showing this approach is more practical when used with young fish.

Used in this study: the Ao Absorbance Microplate Reader from Azure Biosystems

The Ao Absorbance Microplate Reader used by the researchers at the Asian Institute of Technology includes an 8-position filter wheel for the versatility to measure absorbance of many common assays, including TMB (as in the recent work discussed here), Lowry assays, Bradford assays, and more. This plate reader includes a built-in shaker with speed selection, single and dual-read modes, and analysis software to make performance and interpretation of ELISAs and other 96-well plate-based assays fast and easy. Learn more about the Ao Absorbance Microplate Reader by clicking here.

  1. Cain K. Vaccines may be the biggest tool in the fish health toolbox. Aquaculture North Ameriican website. Published March 22, 2021. Accessed January 19, 2024.
  2. Vinh NT, Dong HT, Lan NGT, et al. Immunological response of 35 and 42 days old Asian seabass (Lates calcarifer, Bloch 1790) fry following immersion immunization with Streptococcus iniae heat-killed vaccine. Fish Shellfish Immunol. 2023;138:108802.

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