Elsevier

Fish & Shellfish Immunology

Volume 94, November 2019, Pages 249-257
Fish & Shellfish Immunology

Full length article
Digital gene expression analysis in the liver of ScpB-vaccinated and Streptococcus agalactiae-challenged Nile tilapia

https://doi.org/10.1016/j.fsi.2019.08.072Get rights and content

Highlights

  • DGE technology was applied to detect the gene expression profile of Nile tilapia liver in response to ScpB- vaccinated.

  • A total of 1234 significant differentially expressed unigenes were detected (P<0.05).

  • The identified genes could be categorized into 67 functional groups and mapped to 153 signaling pathways.

  • A complex network including immune and related metabolic pathways in liver of tilapia under ScpB-vaccinated was revealed.

Abstract

In recent years, streptococcal diseases have severely threatened the development of tilapia aquaculture, but effective prevention and control methods have not yet been established. To understand the immune responses of vaccinated Nile tilapia (Oreochromis niloticus), digital gene expression (DGE) technology was applied in this study to detect the gene expression profile of the Nile tilapia (O. niloticus) liver in response to ScpB (Streptococcal C5a peptidase from group B Streptococcus, ScpB) vaccination and a Streptococcus agalactiae-challenge. The control and the ScpB-vaccinated Nile tilapia yielded a total of 25,788,734 and 27,088,598 clean reads, respectively. A total of 1234 significant differentially expressed unigenes were detected (P < 0.05), of which 236 were significantly up-regulated, and 269 were significantly down-regulated (P < 0.05, |fold|>2, FDR<0.05). Of the differentially expressed gene, the identified genes which were enriched using databases of GO and KEGG could be categorized into a total of 67 functional groups and were mapped to 153 signaling pathways including 15 immune-related pathways. The differentially expressed genes (TLR1, TLR2, TLR3, TLR5, TLR9, MyD88, C3, IL-1β, IL-10) were detected in the expression profiles, and this was subsequently verified via quantitative real-time PCR (qPCR). The results of this study can serve as a basis for future research not only on the molecular mechanism of S. agalactiae invasion, but also on the anti-S. agalactiae mechanism in targeted tissues of Nile tilapia.

Introduction

Streptococcus agalactiae, also known as group B streptococcus (GBS), is a gram-positive intracellular bacterium. It was first reported in 1939 as an important opportunistic agent [1]. Over the past few decades, the bacterium was recognized as a major cause of zoonosis, with a broad host range including humans, mice (Mus musculus), cattle (Bos taurus), cats (Felis catus), dogs (Canis lupus familiaris), camels (Camelus bactrianus), horses (Equus caballus), pigs (Sus scrofa), and fish [2]. Among fish species, tilapia (Oreochromis niloticus) is very sensitive to S. agalactiae, so continuous outbreaks of S. agalactiae infection have been reported in tilapia farms throughout the world. Tilapia is an important fish for the economy, especially in China. However, infectious diseases caused by S. agalactiae have been severe in recent years, resulting in great economic losses [3]. S. agalactiae mainly invade the tilapia brain, liver, spleen, and kidney, causing typical symptoms such as anorexia, erratic swimming, exophthalmia, corneal opacity, and hepatomegaly, leading to high mortalities in infected fish [4].

Vaccination is the most environmentally friendly disease control strategy and one of the most effective methods of combating threatening diseases in fish [5]. A number of vaccines are currently commercially available for use in the aquaculture industry, including the live attenuated Edwardsiella ictaluri vaccine for use in catfish (Ictalurus punctatus) [6] and the bacterin and polyvalent bacterin vaccines used against pathogenic vibriosis (Vibrio anguillarum and Vibrio ordalii) [7], furunculosis (Aeromonas salmonicida) [7,8], and enteric red mouth disease (Yersinia ruckeri), and new vaccines continue to be developed [[9], [10], [11]]. Although vaccines are expected to induce long-term, lymphocyte-mediated immunoprotection in fish, little is known about the basic protective mechanisms elicited by the immunization of fish. Moreover, studies on the fish antibacterial system and on the interactive mechanism or immune responses between host cells and S. agalactiae are still in their infancy.

Although many studies have investigated the molecular responses of tilapia against S. agalactiae infection, most of them focused on the characterization of the expression profiles of certain genes by qPCR [[12], [13], [14], [15], [16]]. Transcriptome profiling is a powerful method for analyzing gene products in cells or tissues. Newly developed deep sequencing methods, such as Solexa/Illumina RNA-seq and DGE, have dominated transcriptome studies. Research using these methods has already altered our view of the extent and complexity of eukaryotic transcriptomes, facilitated the discovery of novel genes, and stimulated comparative and integrative genomics. These transcriptomic methods have also been used on tilapia with the S. agalactiae infection [2,17,18]. Studies on these methods mainly focused on the gene transcript changes in the spleen and kidney of tilapia before and after S. agalactiae infection occurs. Many immune-related genes and signaling pathways were found, both of which represent an important anti-bacterial mechanism in tilapia at the early stage of S. agalactiae infection.

The aim of this study was to use deep-sequencing methods to further investigate the DGE profile in the liver of ScpB (Streptococcal C5a peptidase from group B Streptococcus, ScpB)-vaccinated and S. agalactiae-challenged Nile tilapia. Our findings may serve as a basis for future research not only on the molecular mechanism of S. agalactiae invasion, but also on the anti-S. agalactiae mechanism in the targeted tissues of Nile tilapia.

Section snippets

Bacterial strains and growth conditions

S. agalactiae ZP-N was isolated from Nile tilapia (O. niloticus) and preserved in the Pearl River Fisheries Research Institute at the Chinese Academy of Fishery Science. Escherichia coli DH5α and BL21 (DE3) were purchased from Takara, Dalian, China. The S. agalactiae ZP-N strain was cultured in a brain-heart infusion broth (BHI, Huankai Co Ltd., Guangzhou, China) at 28 °C, whereas the E. coli strains were cultured in a Luria-Bertani (LB) broth medium at 37 °C.

Experimental fish

Healthy Nile tilapia (19.5 ± 3.2 g)

ScpB vaccination and S. agalactiae challenge

Two days after the S. agalactiae challenge, mortalities were observed in the PBS-vaccinated control group without clinical signs. The mortalities in the control group continued until 14 days post-challenge. Clinical signs of S. agalactiae infection, which included anorexia, erratic swimming, exophthalmia, and corneal opacity, manifested on day 5 post-challenge. The cumulative mortality rate reached 62.82% in the control group challenged with four times LD50 (8.4 × 107 CFU/mL). In the

Discussion

Many studies have investigated gene and gene expression changes in naïve infected fish [[26], [27], [28]], in fish re-infected after survival from a previous infection [29], or in fish that differed in their inherent disease susceptibility. Some other studies also have looked at the effects of vaccination, with a primary focus on events occurring immediately after vaccination [[30], [31], [32], [33]], but few studies have focused on the early events that occur post-challenge of vaccinated fish.

Conclusions

In summary, a total of 1234 significant differentially expressed unigenes were detected (P < 0.05) in the tilapia liver between the ScpB-vaccinated and the PBS sham-vaccinated control at 48 h after S. agalactiae challenged, of which 236 were significantly up-regulated, and 269 were significantly down-regulated (P < 0.05, |fold|>2, FDR<0.05). Of the differentially expressed gene, the identified genes could be categorized into a total of 67 functional groups and were annotated to 153 signaling

Acknowledgments

This work was financially supported by Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization Open Project of Ministry of Agriculture (KF201309), China Agricultural Research System (No. CARS-46) and Provincial Special Project for Promoting Economic Development (YueNong 2019B8).

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