Exploration and comparison of bacterial communities present in bovine faeces, milk and blood using 16S rRNA metagenomic sequencing

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Authors

Mtshali, Khethiwe
Khumalo, Zamantungwa Thobeka Happiness
Kwenda, Stanford
Arshad, Ismail
Thekisoe, Oriel Matlahane Molifi

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Public Library of Science

Abstract

Cattle by-products like faeces, milk and blood have many uses among rural communities; aiding to facilitate everyday household activities and occasional rituals. Ecologically, the body sites from which they are derived consist of distinct microbial communities forming a complex ecosystem of niches. We aimed to explore and compare the faecal, milk and blood microbiota of cows through 16S rRNA sequencing. All downstream analyses were performed using applications in R Studio (v3.6.1). Alpha-diversity metrics showed significant differences between faeces and blood; faeces and milk; but non-significant between blood and milk using Kruskal-Wallis test, P < 0,05. The beta-diversity metrics on Principal Coordinate Analysis and Non-Metric Dimensional Scaling significantly clustered samples by type (PERMANOVA test, P < 0,05). The overall analysis revealed a total of 30 phyla, 74 classes, 156 orders, 243 families and 408 genera. Firmicutes, Bacteroidota and Proteobacteria were the most abundant phyla overall. A total of 58 genus-level taxa occurred concurrently between the body sites. The important taxa could be categorized into four potentially pathogenic clusters i.e. arthropod-borne; food-borne and zoonotic; mastitogenic; and metritic and abortigenic. A number of taxa were significantly differentially abundant (DA) between sites based on the Wald test implemented in DESeq2 package. Majority of the DA taxa (i.e. Romboutsia, Paeniclostridium, Monoglobus, Akkermansia, Turicibacter, Bacteroides, Candidatus_Saccharimonas, UCG-005 and Prevotellaceae_UCG-004) were significantly enriched in faeces in comparison to milk and blood, except for Anaplasma which was greatly enriched in blood and was in turn the largest microbial genus in the entire analysis. This study provides insights into the microbial community composition of the sampled body sites and its extent of overlapping. It further highlights the potential risk of disease occurrence and transmission between the animals and the community of Waaihoek in KwaZulu-Natal, Republic of South Africa pertaining to their unsanitary practices associated with the use of cattle by-products.

Description

DATA AVAILABILITY STATEMENT : All FastQ sequence files generated from this work are available from the National Center for Biotechnology Information’s Short Reads Archive, under BioProject number PRJNA777568, Accession numbers SRI168760 -SRI168784. All relevant data are within the manuscript and its Supporting Information files.
SUPPLEMENTARY MATERIAL : S1 Fig. A: Alpha diversity box-plots showing Chao1 richness estimates per sample group. *Significant at P < 0,05. B: Alpha diversity box-plots showing Shannon diversity estimates per sample group. *Significant at P < 0,05. C: Alpha diversity box-plots showing Simpson’s diversity estimates per sample group. *Significant at P < 0,05. https://doi.org/10.1371/journal.pone.0273799.s001
S2 Fig. UpSetR intersection plot showing number of unique and shared taxa at family level between faeces, milk and blood groups. https://doi.org/10.1371/journal.pone.0273799.s002
S1 Table. Read counts tracked through the DADA2 pipeline including ASV counts, richness and genus level-resolved ASVs per sample. https://doi.org/10.1371/journal.pone.0273799.s003
S2 Table. Alpha diversity values calculated using Shannon, Simpson and Chao1 indices. https://doi.org/10.1371/journal.pone.0273799.s004
S3 Table. Total number of taxa detected per taxonomic rank across bovine faeces, milk and blood. https://doi.org/10.1371/journal.pone.0273799.s005
S4 Table. Top 15 abundant taxa with their respective overall rankings and distribution across the three sample groups. https://doi.org/10.1371/journal.pone.0273799.s006
S5 Table. Prevalence of potentially pathogenic genera of veterinary significance per sample group. https://doi.org/10.1371/journal.pone.0273799.s007
S6 Table. Bacterial taxa shared between bovine faeces, milk and blood and their overall raw and relative abundances. https://doi.org/10.1371/journal.pone.0273799.s008
S7 Table. Genus-level taxa exclusively detected and shared between faeces, milk and blood samples. https://doi.org/10.1371/journal.pone.0273799.s009
S8 Table. A-C Differentially abundant taxa between blood and faeces; blood and milk and; faeces and milk (Padj < 0,01). https://doi.org/10.1371/journal.pone.0273799.s010
S1 Raw images. Gel electrophoresis image of Anaplasma PCR targeting the 16S rRNA gene from blood samples. Image taken under UV transillumination using Enduro™ GOS gel documentation system. Lane 1 = 1 kb DNA ladder; 2–10 = Anaplasma positive samples; 11 = nuclease free H20 (-ve); 12 = A. marginale (+ve). https://doi.org/10.1371/journal.pone.0273799.s011

Keywords

Cattle by-products, Bovine faeces, Milk, 16S rRNA metagenomic sequencing, Blood microbiota

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Citation

Mtshali, K., Khumalo, Z.T.H., Kwenda, S., Arshad, I. & Thekisoe, O.M.M. (2022) Exploration and comparison of bacterial communities present in bovine faeces, milk and blood using 16S rRNA metagenomic sequencing. PLoS One 17(8): e0273799. https://doi.org/10.1371/journal.pone.0273799.