Co-Occurrence of Francisella, Spotted Fever Group Rickettsia, and Midichloria in Avian-Associated Hyalomma rufipes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Trapping of Birds and Collection of Ticks
2.2. DNA Extraction
2.3. Molecular Screening and Confirmation Analyses
2.3.1. Francisella
2.3.2. Spotted Fever Group Rickettsia
2.4. Tick Taxon
2.5. Characterization
2.5.1. Sanger Sequencing
2.5.2. Spotted Fever Group Rickettsia
2.5.3. Metagenomic Sequencing
2.5.4. Enrichment
2.5.5. Taxonomic Classification of Sequence Reads
2.5.6. Genome Assembly
Enriched Samples
Non-Enriched Samples
2.5.7. Tick Species Confirmation
2.5.8. Phylogenetic Analyses
Tick Phylogenies
Whole Genome and Mitochondrion Phylogenies
2.6. Genome Analysis
2.6.1. Average Nucleotide Identity
2.6.2. Biotin Synthesis Pathways
3. Results
3.1. Bird Trapping and Tick Collection
3.2. Tick Determination
3.3. Detection and Determination
3.3.1. Francisella
3.3.2. Spotted Fever Group Rickettsia
3.3.3. Co-Occurrence
3.3.4. Metagenomics
3.3.5. Phylogenetic Inference of Metagenome-Assembled Genomes
3.4. Genome Analyses
3.4.1. Average Nucleotide Identity
3.4.2. Biotin Gene Conservation
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Organism | Genus/Group/ Species | Target Gene | PCR ID | Name | Sequence (5′ → 3′) | Amplicon (bp) | Reference |
---|---|---|---|---|---|---|---|
Tick | 12S rDNA | 12S | T1B | AAA CTA GGA TTA GAT ACC CT | 320 | [43] | |
T2A | AAT GAG AGC GAC GGG CGA TGT | ||||||
Francisella | fopA | Forward | GGC AAA TCT AGC AGG TCA AGC | [40] | |||
Francisella | FopA | Reverse | CAA CAC TTG CTT GAA CAT TTC TAG | 89 | |||
Probe | GGT GCT TGG GAT GTG GGT GGT G | ||||||
sucC | Forward | AAC TGG CTG ACC TTC AGC AT | [41] | ||||
Francisella | GF1 | Reverse | GTG GTC GTG GTA AAG CTG GT | 125 | |||
Probe | CCG ATT AGG CTT TCT GCT ACT TCA CGA | ||||||
lpnA | Tul4 | Forward | ACC CAC AAG GAA GTG TAA GAT TA | 76 | [40] | ||
F. tularensis | Reverse | GTA ATT GGG AAG CTT GTA TCA TG | |||||
Probe | AAT GGC AGG CTC CAG AAG GTT CTA AGT | ||||||
Forward | CGC AGG TTT AGC GAG CTG TT | ||||||
F. tularensis | lpnA | iQFt1 | Reverse | GCA GCT TGC TCA GTA GTA GCT GTC T | 108 | [42] | |
Probe | CAT CAT CAG AGC CAC CTA ACC CTA | ||||||
Rickettsia | SFG | gltA | GltA | SFG_gltA_F | CCT TTT GTA GCT CTT CTC ATC C | 145 | [40] |
SFG_gltA_R | GCG ATG GTA GGT ATC TTA GCA A | ||||||
SFG_gltA_P | TGG CTA TTA TGC TTG CGG CTG TCG GT | ||||||
Rickettsia | 17 kDa | 17 kDa | Rr17 kDa.61p | GCT CTT GCA AC TTC TAT GTT | 434 | [45] | |
Rr17 kDa.492n | CAT TGT TCG TCA GGT TGG CG |
Method | Microfluidic qPCR (Screening) | qPCR (Confirmation) | ||||
---|---|---|---|---|---|---|
Species/Genus | F. tularensis | Francisella | F. tularensis | Francisella | ||
Tick/PCR ID | Tul4 | FopA | iQFt1 | Tul4 | GF1 | FopA |
D14IT15.2 | Positive Ct = 26.9 | Positive Ct = 22.3 | Negative Ct = N/A | Negative Ct = N/A | Positive Ct = 28.2 | Positive Ct = 29.6 |
D14IT20 | Positive Ct = 26.6 | Positive Ct = 21.6 | Negative Ct = N/A | Negative Ct = N/A | Positive Ct = 19.8 | Positive Ct = 29.6 |
Tick ID | D14IT15.2 | D14IT20 | ||
---|---|---|---|---|
Total Reads (M) | 225.9 | 173.6 | ||
Genus/Species | Reads (M) | % | Reads (M) | % |
Rickettsia | 44.4 | 19.7 | 103.5 | 59.6 |
R. rhipicephali | 38.8 | 17.2 | 93.2 | 53.7 |
Midichloria | 9.4 | 4.2 | 0.95 | 0.55 |
M. mitochondrii | 9.4 | 4.2 | 0.95 | 0.55 |
Francisella | 0.080 | 0.0035 | 0.026 | 0.015 |
FLE | 0.074 | 0.0035 | 0.025 | 0.014 |
Tick ID | D14IT15.2 | D14IT20 | ||
---|---|---|---|---|
Organism | ANIb (%) | Genome | ANIb (%) | Genome |
FLE-Om | 96.8/97.0 | MAG | 96.7/96.9 | MAG |
(GCF_002095075) | ||||
Rickettsia rhipicephali1 (R. aeschlimannii 2) | 99.8/99.8 | MAG | 99.8/99.9 | MAG |
(GCA_001051325) | ||||
Midichloria mitochondrii | 91.5/91.7 | MAG | 91.8/92.3 | MAG |
(GCA_000219355) | ||||
Hyalomma rufipes | 98.1/98.1 | MAG 3 | 98.0/98.1 | MAG 3 |
(KY457528) |
Enriched | Non-Enriched | ||||
---|---|---|---|---|---|
Homolog in Midichloria/Francisella Genomes | D14IT15.2 | D14IT20 | D14IT15.2 | D14IT20 | Genes Involved in the Biotin Synthesis |
lcl|NC_015722.1_cds_WP_013950979.1_473 [M. mitochondrii] | 0 * | 0 | 100 | 100 | bioA |
lcl|NC_015722.1_cds_WP_013950663.1_135 [M. mitochondrii] | 13.4 | 0 | 99.0 | 98.6 | bioB |
lcl|NC_015722.1_cds_WP_237697388.1_131 [M. mitochondrii] | 0 | 0 | 99.7 | 95.4 | bioC |
lcl|NC_015722.1_cds_WP_013950658.1_130 [M. mitochondrii] | 0 | 0 | 93.7 | 93.5 | bioD |
lcl|NC_015722.1_cds_WP_237697389.1_134 [M. mitochondrii] | 0 | 0 | 88.0 | 87.9 | bioF |
lcl|NZ_CP013022.1_cds_WP_064461154.1_1224 [F. persica] | 21.5 | 19.7 | 0 | 0 | Homolog to bioA in F. tularensis (FTT_0938) |
lcl|NZ_CP013022.1_cds_WP_064461748.1_1225 [F. persica] | 100 | 100 | 0 | 0 | Homolog to bioB in F. tularensis (FTT_0937c) |
lcl|NZ_CP013022.1_cds_WP_064461156.1_1227 [F. persica] | 76.3 | 74.3 | 0 | 0 | Homolog to bioC in F. tularensis (FTT_0935c) |
lcl|NZ_CP013022.1_cds_WP_064461157.1_1228 [F. persica] | 99.3 | 99.3 | 0 | 32.5 | Homolog to bioD in F. tularensis (FTT_0934c) |
lcl|NZ_CP013022.1_cds_WP_064461155.1_1226 [F. persica] | 63.3 | 64.4 | 0 | 0 | Homolog to bioF in F. tularensis (FTT_0936c) |
Reads deposited to NCBI sequence read archive | SRR16203935 | SRR16203936 | SRR16203939 | SRR16203940 |
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Hoffman, T.; Sjödin, A.; Öhrman, C.; Karlsson, L.; McDonough, R.F.; Sahl, J.W.; Birdsell, D.; Wagner, D.M.; Carra, L.G.; Wilhelmsson, P.; et al. Co-Occurrence of Francisella, Spotted Fever Group Rickettsia, and Midichloria in Avian-Associated Hyalomma rufipes. Microorganisms 2022, 10, 1393. https://doi.org/10.3390/microorganisms10071393
Hoffman T, Sjödin A, Öhrman C, Karlsson L, McDonough RF, Sahl JW, Birdsell D, Wagner DM, Carra LG, Wilhelmsson P, et al. Co-Occurrence of Francisella, Spotted Fever Group Rickettsia, and Midichloria in Avian-Associated Hyalomma rufipes. Microorganisms. 2022; 10(7):1393. https://doi.org/10.3390/microorganisms10071393
Chicago/Turabian StyleHoffman, Tove, Andreas Sjödin, Caroline Öhrman, Linda Karlsson, Ryelan Francis McDonough, Jason W. Sahl, Dawn Birdsell, David M. Wagner, Laura G. Carra, Peter Wilhelmsson, and et al. 2022. "Co-Occurrence of Francisella, Spotted Fever Group Rickettsia, and Midichloria in Avian-Associated Hyalomma rufipes" Microorganisms 10, no. 7: 1393. https://doi.org/10.3390/microorganisms10071393