Abstract

Background

The gut is primarily responsible for digestion and nutrient absorption, plays essential roles in immune regulation and metabolic balance, and is supported by a diverse microbiome essential for digestion, absorption, and defence from pathogens. Understanding gut physiology and pathophysiology in pre-weaned calves is essential, as infections like cryptosporidiosis can lead to gut dysbiosis, impair growth, and negatively affect long-term productivity. Faeces are considered easily accessible biological specimens that can be used to monitor gastrointestinal disorders. The methods employed in this study aimed to investigate the potential use of faecal extracellular vesicles (fEVs) as a non-invasive tool for assessing gut health and infections in calves. Particularly, considering Cryptosporidiosis as a model for gut infectious disease.

Results

The analysis using a hybrid reference-based metaproteomic approach revealed that the proteomic profiles of fEVs significantly differed from that of faecal crude (FC) suspensions. Both sample types contained microbial and host proteins, which are important for maintaining gut defence and microbial homeostasis. However,Cryptosporidiumspp. infection significantly shifted the fEV proteome, reducing both host and microbial proteins involved in gut defence. It also reduced proteins from microbes that are important for maintaining microbial homeostasis, while increasing stress-related proteins. Further, lyophilisation of fEVs significantly altered the protein profiles.

Conclusion

These findings underscore that fEVs contain host and microbial proteins that are a valuable resource for studying gut physiology, pathophysiology, host-microbe-pathogen interactions, and microbiome dynamics. Changes in the proteomic profile of fEVs duringCryptosporidiumspp. infection demonstrates the pathogen’s ability to manipulate host immune defences and microbiome composition for its survival and replication. Overall, these findings support the utility of fEV proteomics as a non-invasive platform for biomarker discovery and advancing research in gastrointestinal health and disease in livestock.

Data Availability

Mass spectrometry data are available in ProteomeXchange Consortium via the PRIDE with the dataset identifier PXD068377. The additional data that support the findings of this study are available from the first and corresponding author, upon reasonable request.

Abbreviations

  • BCV:: Bovine coronavirus
  • BP:: Biological processes
  • CC:: Cellular component
  • EVs:: Extracellular vesicles
  • FC:: Faecal crude
  • fEV-H:: Faecal extracellular vesicles from healthy calves
  • fEV-I:: Faecal extracellular vesicles from Cryptosporidium spp. infected calves
  • fEV-L:: Lyophilized faecal extracellular vesicles
  • fEV-NL:: Non-lyophilized faecal extracellular vesicles
  • fEVs:: Faecal extracellular vesicles
  • GI:: Gastrointestinal
  • GO:: Gene Ontology
  • LC–MS/MS:: Liquid chromatography/mass spectrometry-mass spectrometry
  • MF:: Molecular function
  • NTA:: Nanoparticle tracking analysis
  • PBS:: Dulbecco’s phosphate-buffered saline
  • PCA:: Principal component analysis
  • SEC:: Size exclusion chromatograph
  • TEM:: Transmission electron microscopy
  • VSN:: Variance stabilizing normalization
  • WB:: Western blotting

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Acknowledgements

The authors would like to thank the following people, (I) Professor Ester Oras, Dr. Rémi Philippe Barbieri from the ARCHEMY LAB, Institute of Chemistry, University of Tartu, Estonia; (II) Dr. James Ord, Faculty of Biological and Environmental Sciences, Organismal and Evolutionary Biology Research Programme and Viikki Plant Science Centre, University of Helsinki, Finland; and (III) Dr. Terhi Ruuska-Loewald, Dr. Anna Kaisanlahti and Professor Justus Reunanen, Biocenter Oulu, University of Oulu, Oulu, Finland for their invaluable mentorship, particularly in proteomics data analysis. We also express our sincere gratitude to Dr. Getnet Balcha Midekessa from the Estonian University of Life Sciences, Estonia, for arranging the sample lyophilisation facility and providing essential technical assistance.

Funding

This project was funded by the European Union through HORIZON 2020 research and innovation program under grant agreement No 857418 "COMBIVET", HORIZON Coordination and Support Actions under grant agreement No 101079349 “OH-Boost", Co-funded by the European Union's Horizon Europe Project 101136346 EUPAHW. Views and opinions expressed are however, those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. Supported by the Estonian University of Life Sciences base funding project (P17019VLKM).

Ethics Declaration

Ethics approval and consent to participate

All experimental protocols involving animals in this study were reviewed and approved by the Ethical Committee of Animal Experiments of the Estonian Ministry of Agriculture (animal project permit No. 116).

Consent for publication

All authors have approved the final manuscript for publication.

Competing interests

The authors declare no competing interests.

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