﻿***Title of the dataset / Data underlying the publication: ‘Effects of E. coli Nissle 1917 on the Porcine Gut Microbiota and Immune System in Early Life’***

***Creators***
Mirelle Geervliet 1,†, Hugo de Vries 2,3,†, Christine A. Jansen 1,4, Victor P.M.G. Rutten 4,5, Hubèrt van Hees 6, Caifang Wen 3, 
Kerstin Skovgaard 7, Giacomo Antonello 8, Huub F.J. Savelkoul 1, Hauke Smidt 3, Edwin Tijhaar 1,†, and Jerry M. Wells2,†,*

1	Cell Biology and Immunology Group, Wageningen University & Research, Wageningen, The Netherlands; mirelle.geervliet@wur.nl; huub.savelkoul@wur.nl; edwin.tijhaar@wur.nl; christine.jansen@wur.nl 
2	Host-Microbe Interactomics Group, Wageningen University & Research, Wageningen, The Netherlands; jerry.wells@wur.nl
3	Laboratory of Microbiology, Wageningen University & Research, Wageningen, The Netherlands; hugo.devries@wur.nl; caifang.wen@wur.nl; hauke.smidt@wur.nl 
4	Department of Biomolecular Health Sciences, Division of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands; v.rutten@uu.nl 
5	Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa; v.rutten@uu.nl 
6	Research and Development, Trouw Nutrition, Amersfoort, The Netherlands; hubert.van.hees@trouwnutrition.com
7       Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark; kesk@dtu.dk
8       Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy; giacomo.antonello@unitn.it

* Correspondence: jerry.wells@wur.nl

† These authors have contributed equally to this work and share first or last authorship.

***Related publication***
Title: Effects of E. coli Nissle 1917 on the Porcine Gut Microbiota and Immune System in Early Life
DOI: 10.3389/fcimb.2022.842437
Received: December 23th, 2021; Accepted: February 1st, 2022; Published: 2022

***General Introduction***
This README file corresponds to the data corresponding to Figure 6 of the manuscript.
This dataset contains data collected during an in vivo experiment with pigs at the Wageningen University as part of the PhD Thesis Projects of Mirelle Geervliet and Hugo de Vries (first authors of the manuscript). 
This research project was made possible by The Netherlands Organisation for Scientific Research and Vereniging Diervoeders Nederland (VDN).

***Purpose of analysis***
Recognition of EcN by immune cells may induce altered immune responses upon a secondary stimulation, thereby influencing the immune competence of the pig. 
To test this, we assessed if pigs treated with EcN showed an enhanced immune response to a live attenuated Salmonella vaccine (Salmoporc®). 
A clear vaccine induced increase in Salmonella specific antibodies was observed, but EcN did not enhance the vaccination response. 

***Methodological information***
Blood was collected on days 14, 26, 43 and 69 from the jugular vein of the pig using Natrium Heparin tubes (S-monovette®, Sarstedt). 
From every pen, two control animals or two EcN treated animals were randomly selected and followed over time (n = 16 per treatment group).
Blood samples were centrifuged at 2000 x g for 10 min to collect serum. Serum was stored at -20 °C until further use. 
To analyse the antibody response against Salmonella Typhimurium (IgG, IgM or IgA), an in-house ELISA was optimised and performed (for more information on the methodology see manuscript).
The primary Salmoporc vaccination was given on day 21 and the booster on day 45. 

***Data specific information***

R script can be found under the following folder: Data_analyses_in_R_Studio.
This folder also contains the data files used in the subfolder 'input_data'. This includes the metadata file.

In folder 'Figure 6', four subfolders can be found namely:

1. Raw_data:
Three Excel files with raw data (Optical Density or O.D. values) from day 14, day 35 and day 69. 
The raw data is displayed in 96-well plate format (1-12 and A-H). 
Every plate has a specific name corresponding to the type of Immunoglobulin tested. 
At each time point we tested the the presence of Salmoporc specific IgM, IgA and IgG. 
The fourth Excel file 'Processed raw data' contains an Excel file with processed data (Optical Density or O.D. values from day 14, day 35 and day 69) and more specific information (ear tag number and treatment). 
Note that this file contains different sheets (day 14, day 35 and day 69).  

2. Unprocessed_Figure
Contains the unprocessed picture (generated by GraphPad Prism). 

3. Final_Figure
Contains the final version of Figure 6 (as presented in the manuscript). 
Adjustments have been made in Adobe Illustrator. 

4. Excel_File_R
Contains six Excel (csv) files (three of them with log-transformed data) used for the R code to generate graphs and for statistical analysis. 


