Introduction
Streptococcus agalactiae, a Gram-positive pathogenic bacterium also known as group B streptococcus, is recognized as a causative agent of zoonosis (Gauthier, 2015). With a broad host range, it is one of the major causes of infection outbreaks in Nile tilapia (Oreochromis niloticus) with significant morbidity and mortality, resulting in great economic loss and threatening the development of tilapia aquaculture worldwide (Zhang, 2021). This bacterium species can be subdivided into ten serotypes (Ia, Ib, and II–IX) based on its capsular polysaccharide composition (Poyart et al., 2007), and among these serotypes, Ia, Ib, and III are the most frequent in aquatic animals (Li et al., 2003). The infection leads to septicaemia and encephalitis in fish, with noticeable clinical signs including lethargy, erratic swimming, exophthalmia, and ascites (Pretto-Giordano et al., 2010). Our research group recently isolated two distinct serotypes of S. agalactiae in Brazil. The first, E8ang2 (serotype Ib), was isolated in the northern state of Paraná, while the second, Maranhão (serotype III), was isolated in the Maranhão state. Upon further investigation, we noticed different patterns of responses to infection in tilapia with these strains. The Maranhão strain induces significant brain damage, resulting in severe signals of erratic swimming and a higher mortality rate in the initial days of infection. In contrast, E8ang2 kills the same number of animals but more slowly and with a lower incidence of noticeable brain-related symptoms. This study aims to characterize the brain transcriptomes of tilapia after infection with S. agalactiae and understand the reason for the different outcomes by comparing gene expression profiles between animals infected with strains of different serotypes.
Materials and Methods
This study followed the recommendations established by the Animal Ethics Committee, State University of Londrina (process nº CEUA 45/2017), Brazil. Nile tilapia were randomly divided into three groups: i) control, ii) challenged with the E8ang2 strain (serotype Ib), and iii) challenged with the Maranhão strain (serotype III). The fish were anesthetized and intraperitoneally injected with 0.1 mL/fish of bacterial suspension, whereas the control group fish were injected with an equal volume of broth medium. After bacterial exposure, the fish were monitored daily, and animals that showed signs of erratic swimming were removed from the aquaria and killed by medullary section. The brain was collected and immediately frozen in liquid nitrogen. The RNA was extracted, and 14 Illumina libraries were prepared (4 for Control, 5 for E8ang2, and 5 for Maranhão groups) and sequenced as paired-end reads (150-bp). After quality control and trimming of adapters using the fastp software, the reads were mapped to the Nile tilapia genome using HISAT2 and annotated using featureCounts. The differential expression of genes across treatment groups was determined with DESeq2. Enrichment of KEGG pathways and gene ontology were performed in g:Profiler.
Results
In response to exposure to strains of S. agalactiae, several immune response-associated genes were upregulated in the tilapia brain. These genes are mainly related to phagocytosis, cytokine production, and immune cell recruitment, suggesting that the immune response of tilapia to S. agalactiae infection is predominantly pro-inflammatory. However, genes involved in tissue repair and oxygen transport were downregulated. Additionally, the energetic demands of the immune response may affect the expression of genes related to the feeding behavior of tilapia during S. agalactiae infection.
Comparing the results of exposure to both strains, the animals exhibited an increase in the expression of pathogen recognition-related genes and inflammation-associated genes when exposed to the E8ang2 strain. These results suggest that the E8ang2 strain can be readily identified by the immune system triggering a more robust immune response. Furthermore, the upregulation of genes involved in neuroplasticity indicates a possible faster recovery of the injured tissue in the exposure to E8ang2. Meanwhile, the downregulation of some hormone-related genes may indicate a disturbance in regulating hormonal pathways involved in stress responses.
Overall, our results suggest that the two strains of S. agalactiae may elicit different responses from the host immune system and neuroendocrine pathways. The study of this disease holds significant importance for aquaculture and human medicine. Additionally, elucidation of the mechanisms involved in the host–pathogen response is crucial for the success of new therapies. Our research is not only relevant for a better understanding and description of the tilapia immune system, but it also has the potential to provide new insights into the development of prophylactic measures for this species.
References
Gauthier, D. T. (2015). Bacterial zoonoses of fishes: a review and appraisal of evidence for linkages between fish and human infections. The Veterinary Journal, 203(1), 27-35.
Li, L., Wang, R.; Liang, W. Gan, X., Huang, T., Huang, Y., Li, J., Shi, Y., Chen, M., Luo, H. (2013). Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China. Veterinary Microbiology, 167(3-4),719-724.
Poyart, C., Tazi, A., Réglier-Poupet, H., Billoët, A., Tavares, N., Raymond, J., Trieu-Cuot, P. (2007). Multiplex PCR assay for rapid and accurate capsular typing of group B streptococci. J Clin Microbiol, 45(6),1985-8.
Pretto-Giordano, L. G., Müller, E. E., Freitas, J. C. D., Silva, V. G. D. Evaluation on the Pathogenesis of Streptococcus agalactiae in Nile Tilapia (Oreochromis niloticus). (2010). Brazilian Archives of Biology and Technology, 53, 87-92.
Zhang, Z. (2021). Research advances on Tilapia streptococcosis. Pathogens, 10(5), 558.
Acknowledgement
This study was financed by the Research Council of Norway (NORBRAQUA, grant nr. 310103).