Genomic analysis and immune response in a murine mastitis model of vB_EcoM-UFV13, a potential biocontrol agent for use in dairy cows

Abstract

Bovine mastitis remains the main cause of economic losses for dairy farmers. Mammary pathogenic Escherichia coli (MPEC) is related to an acute mastitis and its treatment is still based on the use of antibiotics. In the era of antimicrobial resistance (AMR), bacterial viruses (bacteriophages) present as an efficient treatment or prophylactic option. However, this makes it essential that its genetic structure, stability and interaction with the host immune system be thoroughly characterized. The present study analyzed a novel, broad host-range anti-mastitis agent, the T4virus vB_EcoM-UFV13 in genomic terms, and its activity against a MPEC strain in an experimental E. coli-induced mastitis mouse model. 4,975 Single Nucleotide Polymorphisms (SNPs) were assigned between vB_EcoM-UFV13 and E. coli phage T4 genomes with high impact on coding sequences (CDS) (37.60%) for virion proteins. Phylogenetic trees and genome analysis supported a recent infection mix between vB_EcoM-UFV13 and Shigella phage Shfl2. After a viral stability evaluation (e.g pH and temperature), intramammary administration (MOI 10) resulted in a 10-fold reduction in bacterial load. Furthermore, pro-inflammatory cytokines, such as IL-6 and TNF-α, were observed after viral treatment. This work brings the whole characterization and immune response to vB_EcoM-UFV13, a biocontrol candidate for bovine mastitis.

Figure 1

Genome map of vB_EcoM-UFV13. The linear genome was circularized in order to improve its visualization. CDS, ORF, GC content, GC skew+ and GC skew- are reported in circles from outside inwards.

Figure 2

Variants calling between vB_EcoM-UFV13 and Enterobacteria phage T4 were predicted using SnpEff. Only variants with predicted “high” or “moderate” impact on the protein-coding gene were analyzed and functionally categorized.

Figure 3

Phylogenetic relationship between phage UFV13 and genera belonging to the subfamily Tevenvirinae (T4virus, Cc31virus, S16virus, Js98virus and Sp18virus). vB_EcoM-UFV13 is most closely related to Shigella phage Shfl2 and Yersinia phage PST, and is clearly a member of the T4virus genus.

Figure 4

The stability of vB_EcoM-UFV13 under different conditions was evaluated. (A) Reductions of 100, 82, 4 and 32% of viable particles were observed after incubations, respectively, at pHs 2, 4, 10 and 12. (B) vB_EcoM-UFV13 was able to replicate at 30 and 22 °C with an efficiency of plating of 69 and 56%, corresponding; (C) vB_EcoM-UFV13 was inactivated at 95 °C for 5 min; (D) Osmotic shock changing reduced in 84% viral viability.

Figure 5

Viral stability under anionic and cationic detergents showed that vB_EcoM-UFV13 was sensible in all conditions.

Figure 6

Five different cytokines (IL-6, TNF-α, IL-2, IFN-γ and IL-10) were locally measured. Only IL-10, TNF-α and IL-6 were statistically significant (*p < 0.05; **p < 0.01) and is indicative of a pro-inflammatory pattern after phage treatment. IL-17A and IL-4 levels were not detected by the Cytometric Bead Array kit.

Figure 7

Histological analysis of mammary gland after PBS (A), E. coli 30 (B) and treatment with vB_EcoM-UFV13 (C). In C, neutrophil infiltration (indicated by arrow head) was detected 48 hours after phage treatment using MOI 10. Bars: 100 micrometers.