Elsevier

Fish & Shellfish Immunology

Volume 86, March 2019, Pages 25-34
Fish & Shellfish Immunology

Full length article
Local regulation of immune genes in rainbow trout (Oncorhynchus mykiss) naturally infected with Flavobacterium psychrophilum

https://doi.org/10.1016/j.fsi.2018.11.027Get rights and content

Highlights

  • CD4, CD8, perforin and IFNγ mRNA levels are up-regulated in ulcers upon a F. psychrophilum natural infection.

  • Membrane and secreted IgD mRNA are also up-regulated in ulcerative lesions.

  • No immune response is visible at a transcriptional level in the spleen.

  • A high number of cells expressing IgD and CD3 are present in the inflamed skin.

  • These results point to a relevant role of IgD and CD8+ T cells in the local response to F. psychrophilum.

Abstract

Flavobacterium psychrophilum is the etiological agent of bacterial cold water disease (BCWD), also referred to as rainbow trout fry syndrome (RTFS), a disease with great economic impact in salmonid aquaculture. Despite this, to date, not many studies have analyzed in depth how the immune system is regulated during the course of the disease. In the current study, we have studied the transcription of several immune genes related to T and B cell activity in the skin of rainbow trout (Oncorhynchus mykiss) naturally infected with F. psychrophilum in a farm located in Lake Titicaca (Peru). The levels of expression of these genes were tested and compared to those obtained in asymptomatic and apparently healthy rainbow trout. In the case of symptomatic fish, skin samples containing characteristic ulcerative lesions were taken, as well as skin samples with no lesions. Our results pointed to a significant local up-regulation of IgD, CD4, CD8, perforin and IFNγ within the ulcerative lesions. On the other hand, no differences between the levels of expression of these genes were visible in the spleen. To confirm these results, the distribution of IgD+ and CD3+ cells was studied through immunohistochemical techniques in the ulcerative lesions. Our results demonstrate a strong local response to F. psychrophilum in rainbow trout in which IgD and T cells seem to play a major role.

Introduction

Bacterial cold water disease (BCWD), also referred to as rainbow trout fry syndrome (RTFS), is one of the most important diseases affecting the salmonid aquaculture industry worldwide. The disease is caused by a Gram-negative, rod-like, filamentous, and yellow-pigment-producing bacterium, Flavobacterium psychrophilum [1,2]. This pathogenic microorganism not only affects salmonid species such as rainbow trout (Oncorhynchus mykiss) or coho salmon (Oncorhynchus kisutch), but has also been reported to cause disease in non-salmonid species such as eel (Anguilla japonica and Anguilla anguilla), carp (Cyprinus carpio and Carassius carassius) and tench (Tinca tinca) [3,4]. The most commonly documented clinical signs of BCWD in adult fish include ulcerative lesions on the skin and the musculature, predominantly near the caudal peduncle and on the flank, followed by progressive tissue necrosis [1,5,6]. In fry, however, septicemia, anemia and lethargy have been reported without any external ulcers, with mortality rates ranging up to 90% in rainbow trout [2,6,7].

The first report of BCWD was described in rainbow trout in North America in 1941 [8]. Following this initial report, F. psychrophilum was reported from different geographical areas including Europe (Belgium, Denmark, France, Finland, Germany, Italy, and Spain), Asia (Japan and South Korea), Oceania (Australia) and South America (Chile and Peru) [2,[9], [10], [11], [12]]. It has been revealed that culture density and poor water quality with systems lacking filtering devices can favor the permanence of the pathogen and the appearance of the disease. This is the case of some Chilean fish farms in which F. psychrophilum was declared as the etiological agent of BCWD outbreaks since 2013 [13]. In Peru, continuous mortalities in several rainbow trout farms have also been associated to the presence of F. psychrophilum [14,15]. Many of these F. psychrophilum-associated mortalities were reported in rainbow trout reared in cages in Lake Titicaca, within the region of Puno [14]. According to the Peruvian Ministry of Production, the region of Puno was the main producer of rainbow trout in 2016 with a production of 43,000 tonnes, representing 80% of the national production. Within the region of Puno, Lake Titicaca is the largest freshwater lake in South America localized amongst Peru and Bolivia at 3810 m above sea level with an area of 8559 km2 [16].

Although several antigens have been identified as potential vaccine candidates and different serotypes of F. psychrophilum have been tested as inactivated vaccines, to our knowledge, at the moment, only one commercial vaccine is available to prevent BCWD (ALPHA JECT® IPNV-Flavo 0,025, Pharmaq) in Chile [6,13,17,18]. This scenario has promoted the use of antibiotics to reduce economic losses attributable to this disease in salmonid farming, with the consequent negative impact on the environment and the generation of antibiotic resistances [13,19]. To optimize an efficient control strategy, it seems key to have an in-depth understanding of the immune response elicited by the pathogen in the species targeted, especially at mucosal surfaces such as the skin, one of the tissues preferentially damaged by F. psychrophilum. Recently, our group has demonstrated that the rainbow trout skin is a T cell rich organ by means of transcriptomic analysis, immunohistochemistry and flow cytometry [20]. These T cells are not evenly distributed throughout the skin, but are preferentially accumulated in the most anterior area, close to the gills [20]. Furthermore, these skin T cell responses were locally regulated in response to a bath infection with hemorrhagic septicemia virus (VHSV), revealing a major role of skin T lymphocytes in the local response to pathogens [20]. In the same line, T cell functionality has been the focus of studies dealing with inflammatory skin disorders affecting rainbow trout such as red mark syndrome (RMS) or puffy skin disease (PSD) [21,22]. On the other hand, both IgM+ and IgT+ B cells are also known to be present in salmonid skin and respond to infections [23]. IgD, the other Ig isotype present in rainbow trout, is also actively transcribed in rainbow trout skin [20], however its precise role in immunity remains elusive both in fish and mammals. Despite all these previous data, so far, no studies have focused on analyzing the local transcription of genes related to T and B cells in the skin of rainbow trout infected with F. psychrophilum and this is what we have addressed in the current study. We have sampled skin and spleen from rainbow trout obtained from Lake Titicaca (Peru) with ulcerative lesions in which the presence of F. psychrophilum was confirmed. The levels of expression of several immune genes related to T and B cells were tested and compared to those obtained in asymptomatic and apparently healthy rainbow trout, namely CD4, CD8, Eomes, perforin, interleukin 4/13 (IL4/13), interferon γ (IFNγ), IgM, IgT, IgD and the CK11 chemokine, a chemokine strongly expressed in the skin with a major role in antimicrobial immunity [24]. Since our results pointed to a significant local up-regulation of IgD, CD4, CD8, perforin and IFNγ, the distribution of IgD+ and CD3+ cells was studied through immunohistochemical techniques in the ulcerative lesions. Our results reveal a strong local response to F. psychrophilum in rainbow trout in which IgD and T lymphocytes seem to play a major role.

Section snippets

Fish sampling and description of the area where the outbreak occurred

Asymptomatic (showing no external signs of disease) and symptomatic adult rainbow trout (Oncorhynchus mykiss) with typical disease symptoms of BCWD and length of ∼25 cm were obtained from Lake Titicaca in the region of Puno (Peru) between the months of December 2017 and February 2018, the summer season. The experiments described in this work comply with the Law for the Protection of Domestic and Wild Animals kept in captivity (Law 27265/Title 6/Art. 18) and were previously approved by the

Identification of F. psychrophilum by sequencing

Conventional PCR and gel analysis showed that the primers designed to amplify the 16S rDNA of F. psychrophilum amplified a fragment of 239 bp in all skin samples from the rainbow trout sampled (Fig. 2A). The sequencing of these PCR products obtained allowed us to build a consensus sequence and recognize the bacteria identified as F. psychrophilum, showing a 99% identity of this sequence and that of previously reported F. psychrophilum strains. Although F. psychrophilum 16S rDNA was amplified in

Discussion

Studying the immune response elicited during the course of an infection with a specific pathogen is key for the rational development of effective vaccines. In the case of F. psychrophilum, although some previous studies have focused on analyzing the transcriptional response to the pathogen after experimental infections [31,32], none of these studies were performed with naturally infected rainbow trout. Furthermore, these previous studies determined the transcriptional regulation of immune genes

Acknowledgements

The authors want to thank Lucía González and Laura Fernández for technical assistance. Dr. Erin Bromage (University of Massachusetts Dartmouth, USA) is also acknowledged for supplying the anti-IgD and anti-CD3 antibodies used in this study. This work was supported by the European Research Council (ERC Consolidator Grant 2016 725061 TEMUBLYM). Carlos Távara would like to acknowledge the financial support from the Master Program in Aquatic Animal Health of the UPCH subsidized by Cienciactiva of

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