Composition of bacterial communities in the intestine, skin and spleen of farmed rainbow trout infected with Vibrio anguillarum

Published: 13 July 2022| Version 2 | DOI: 10.17632/6m4jz3ywhc.2
Contributors:
,
,
,
Ekaterina Borvinskaya,
Alina Vasileva

Description

Vibrio anguillarum infection in cultured trout, besides its own harmful effects, can also make the fish more susceptible to other infections, both by swamping the host immune system and by creating the skin lesions that serve as a direct gateway for opportunistic bacteria. Samples of tissue were taken from gut, spleen, and mucus from skin lesions of cage-grown rainbow trout suffering from natural Vibrio infections of varying severity. The fish were caught by a net randomly from four neighboring cages. The animals in these cages were from the same full-sibling family batch, the same age, average weight, and were kept with the same stocking density and feeding regime. In addition, a sample of water was taken to document the planktonic bacteria inhabiting the fish farm at the time of study. Total DNA was isolated from these samples and used to produce v3-v4 SSU rRNA amplicons, which were then sequenced on Illumina MiSeq (2x300 bp) and assembled into 97% identity OTUs. Raw sequencing data deposited at NCBI SRA (project ID PRJNA828372, sequencing run IDs SRR18828104 to SRR18828151) and resulted OTU tables deposited here. Table "OTUs" provides data on the composition of bacterial communities in the spleen, skin and intestines of farmed rainbow trout infected with Vibrio anguillarum (SL-heavy infected, IP-moderately infected, AS-asymptomatic). The "Taxonomy" table contains a decryption of the taxonomy of these unique OTUs. The obtained 16S rRNA gene sequencing dataset allows analyzing bacteria co-colonizing skin and intestine of the infected fishes along with the pathogenic Vibrio anguillarum, as well as generally providing knowledge on the spread of bacteria between various bodily compartments in infected trout.

Files

Steps to reproduce

The fish was caught in August 2020 on a trout farm located in the bay of the White Sea, Northwest Russia. The fish were caught by a net randomly from four neighboring cages. Fish with obvious symptoms of infection and those not showing any outward signs of illness were caught (photographs available in https://doi.org/10.6084/m9.figshare.17169380). Each fish was euthanized in a 0.5 ml/L clove oil emulsion for about 5 min. If there were skin lesions, the mucus was scraped from the affected area with a sterile scalpel and fixed in liquid nitrogen. Then the fish was dissected aseptically, the spleen of the fish was weighed and fixed in liquid nitrogen. The intestine was opened with scissors, the contents and parietal microflora of the anterior and posterior intestines were scraped off with a sterile scalpel, pooled, and fixed in liquid nitrogen. The 10L water samples near the cage were collected and filtered through a sterile 0.22 μm filter cartridge. The filter was frozen and stored in liquid nitrogen until DNA extraction. Total DNA was isolated using a DiaGene kit for DNA isolation (Dia-M). For this, 50 mg of the sample was homogenized in lysis LBT buffer with the addition of 8 μl of proteinase K and incubated for 16 hours at 37 °C. The obtained lysate was centrifuged in an Eppendorf centrifuge at 12000 g for 20 minutes. A sorption buffer (1:1.4 v/v) and chloroform (1:1 v/v) were added to the supernatant and centrifuged at 12000 g for 20 minutes. Then, the aqueous phase containing DNA was collected and transferred into DiaGene microcolumns (Dia-M). The columns were washed once with WB1 buffer and twice with WB2 buffer. DNA from the columns was eluted with sterile deionized water. V3–V4 variable regions of the 16S rRNA gene were amplified using the "16S Metagenomic Sequencing Library Preparation" protocol (Part # 15044223 Rev. B; Illumina), with the primer pair 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC. After obtaining the amplicons, the libraries were cleaned and mixed equimolarly using a SequalPrep™ Normalization Plate Kit (ThermoFisher). The resulting pool of libraries was sequenced on an Illumina MiSeq (2x300 bp paired-end reads) with a MiSeq Reagent Kit v3 (600 cycles). The PhiX phage library was used to control the sequencing parameters. Most of the reads related to phage DNA were removed during the demultiplexing process. The obtained paired-end Illumina MiSeq reads were assembled into contigs. After deleting the low-quality reads, unassembled pairs (contigs above 470 nucleotides in length) and chimeric reads, contigs were mapped to SILVA v138.1 reference SSU rRNA alignment. Raw sequencing data were deposited at NCBI SRA (project ID PRJNA828372). Sequences that aligned correctly were used to generate 97% identity OTUs. OTUs that have no more than one read in all libraries were excluded as singletons. All read analyses were performed in mothur 1.44.11 software.

Institutions

Petrozavodskij Gosudarstvennyj Universitet

Categories

Metabarcoding

License