Development of a Multiplex Droplet Digital PCR Assay for the Detection of Babesia, Bartonella, and Borrelia Species
Abstract
:1. Introduction
2. Methods
2.1. Sample Reference Types and DNA Extraction
2.2. Mammalian Cell Line In Vitro Infection
2.3. Experimentally Infected Mouse, Rabbit and Hamster Tissue
2.4. Spiked Blood Samples
2.5. Previously Characterized Borrelia spp. DNA Samples
2.6. DNA Extraction
3. Molecular Methods
3.1. Housekeeping Gene DNA
3.2. Primers and Probes for Bartonella, Borrelia, and Babesia DNA Detection
3.3. DNA Amplification
4. Results
4.1. Naive Blood, Tissue and In Vitro Cultivated Samples
4.2. Detection of Babesia, Theileria and Cytauxzoon Species
Clinical Samples from Naturally and Experimentally Infected Animals
4.3. Detection of Bartonella spp.
4.4. Detection of Borrelia spp.
4.5. Spiked Naïve DNA Blood Samples
4.6. Previously Characterized Borrelia spp. DNA Samples
4.7. Tissues from Experimentally Infected Mice and Cell Lines and from Naturally Infected Vectors
4.8. Dual Detection of Bartonella and Borrelia spp.
4.9. Dual Detection of Bartonella and Babesia
4.10. Babesia, Bartonella, and Borrelia Spiked Naïve Blood Samples
5. Discussion and Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Disclosure
References
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Piroplasma | Sample Type | Host/s | Infection Type |
---|---|---|---|
B. bicornis | Blood | Rhinocerous | Naturally |
B. canis canis | Blood | Dogs | Naturally |
B. canis vogeli | Blood | Dogs | Naturally |
B. coco | Blood | Dogs, Horse | Naturally |
B. conrade | Blood | Dogs | Naturally |
B. divergens | Blood | Rabbit | Experimentally |
B. duncani | Blood | Hamster | Experimentally |
B. felis | Blood | Cats | Naturally |
B. gibsoni | Blood | Dogs | Naturally |
B. gibsoni + B. vulpes | Blood | Dog | Naturally |
B. lengau | Blood | Cats | Naturally |
B. microti | Blood | Hamster | Experimentally |
B. odoncoilei | Blood | Deer, Reindeer | Naturally |
B. vulpes | Blood | Dogs, Foxes | Naturally |
B. vulpes + Babesia spp. | Blood | Raccoon | Naturally |
Babesia spp. | Blood | Dog | Naturally |
Babesia spp. AJB 2006 | Blood | Bear | Naturally |
Babesia spp. NC08 | Blood | Bear | Naturally |
Babesia spp. NC13 | Blood | Bear | Naturally |
Babesia spp. R-214 | Blood | Racoon | Naturally |
Babesia spp. R431 | Blood | Racoon | Naturally |
Babesia spp. R-9879 | Blood | Racoon | Naturally |
Babesia spp. R-CO8 | Blood | Racoon | Naturally |
Babesia spp. SK04 | Blood | Skunk | Naturally |
Babesia spp. SK08 | Blood | Skunk | Naturally |
Babesia spp. WR3 | Blood | Woodrat | Naturally |
C. felis | Blood | Cats | Naturally |
Maned wolf Babesia | Blood | Dogs, Racoons | Naturally |
T. cervi | Blood | Deer, Bears | Naturally |
T. equi | Blood | Horses | Naturally |
Bartonella | Sample Type | Host/s | Infection Type |
---|---|---|---|
B. alsatica | Blood | Rabbits | Naturally |
B. bovis | Blood | Cows | Naturally |
B. chomelii | Blood | Elk | Naturally |
B. clarridgeiae | Blood | Cats, Dogs | Naturally |
B. henselae | Blood | Humans, Cats, Dogs | Naturally |
B. henselae | Tissue (Brain) | Dolphins | Naturally |
B. henselae | Tissue (Body) | Fleas, Sanflies | Naturally |
B. henselae | Tissue (Skin) | Mice | Experimentally |
B. henselae | Tissue (Body) | Tick (I. scapularis) | Naturally |
B. henselae H1 | Blood | Human, Cat, Dog | Spiked |
B. henselae SA2 | Blood | Human, Dogs, Birds | Naturally |
B. henselae SA2 | Blood | Human, Dog | Spiked |
B. henselae SA2 | DH82 cell-line | Dog | Experimentally |
B. henselae SA2 | MCF10A cell-line | Human | Experimentally |
B. koehlerae | Blood | Human, Dog | Spiked |
B. melophagi | Blood | Human, Dog | Spiked |
B. melophagi | Blood | Sheep | Naturally |
B. quintana | Blood | Human, Dog | Spiked |
B. quintana | Blood | Human, Monkey | Naturally |
B. rochalimae | Blood | Dogs | Naturally |
B. tamiae | Blood | Human | Spiked |
B. tamiae | Blood | Woodrats | Naturally |
B. tribocorum | Blood | Rats | Naturally |
B. v. berkhoffii TI | Blood | Human, Dog | Spiked |
B. v. berkhoffii TII | Blood | Human, Dog | Spiked |
B. v. berkhoffii TII | Blood | Human, Coyotes, Foxes | Naturally |
B. v. berkhoffii TIII | Blood | Human, Dog | Spiked |
B. v. berkhoffii TIII | Blood | Skunk | Naturally |
B. volans | Tissue (Heart) | River otter | Naturally |
Bartonella spp. | Blood | Mongooses | Naturally |
Bartonella spp. Rc | Blood | Racoons | Naturally |
Borrelia | Sample Type | Host | Infection Type |
---|---|---|---|
B. afzelii | Blood | Dog | Spiked |
B. bissetii | Tissue (Body) | Tick (I. affinis) | Naturally |
B. burgdorferi | Tissue (Body) | Tick (I. scapularis) | Naturally |
B. burgdorferi B31 | Blood | Human, Dog | Spiked |
B. burgdorferi B31 | DH82 cell line | Dog | Experimentally |
B. burgdorferi B31 | MCF10A cell line | Human | Experimentally |
B. burgdorferi B31 | Tissue (Skin) | Mice | Experimentally |
B. coreaceae | Blood | Dog | Spiked |
B. garinii | Blood | Dog | Spiked |
B. hermsii | Blood | Dog | Spiked |
B. lusitanae | Blood | Dog | Spiked |
B. turicatae | Blood | Dog | Spiked |
Co-Infection | Sample Type | Host | Infection Type |
---|---|---|---|
B. burgdorferi + B. gibsonii | Blood | Dog | Naturally + Spiked |
B. burgdorferi + B. gibsonii | Blood | Dog | Naturally + Spiked |
B. burgdorferi + B. microti | Blood | Human | Spiked |
B. henselae + B. burgdorferi | Blood | Dog | Spiked |
B. henselae + B. burgdorferi | Blood | Human | Spiked |
B. henselae + B. burgdorferi | Tissue (Brain) | Human | Experimentally |
B. henselae + B. burgdorferi | Tissue (Body) | Tick (I. affinis) | Naturally |
B. henselae + B. burgdorferi + B. gibsonii | Blood | Dog | Naturally + Spiked |
B. henselae + B. burgdorferi + B. microti | Blood | Human | Spiked |
B. henselae + B. burgdorferi B31 | Tissue (Skin) | Mice | Experimentally |
B. henselae + B. microti | Blood | Human | Spiked |
B. odoncoilei + B. henselae | Tissue (Body) | Tick (I. affinis) | Naturally |
B. quintana + B. burgdorferi | Blood | Dog | Spiked |
B. quintana + B. burgdorferi | Blood | Human | Spiked |
B. v. berkhoffii TII + B. burgdorferi | Blood | Dog | Spiked |
B. v. berkhoffii TII + B. burgdorferi | Blood | Human | Spiked |
B. vinsonii + B. vulpes | Blood | Gray fox | Naturally |
Borrelia sp. | Strain | Conc. Range (Genome Copies per 5 µL Sample) |
---|---|---|
B. afzelii | Pko | 0.1–10,000 |
B. bavariensis | VS116 | 0.1–10,000 |
B. bissettii | Pgeb | 10,000 |
B. burgdorferi | B31, Pbre | 0.1–10,000 |
B. garinii | PBr, Phei, Pla, Pref, PWudII | 0.1–10,000 |
B. hermsii | 0.1–10,000 | |
B. lusitanae | PotiB2 | 0.1–10,000 |
B. miyamotoi | 10,000 | |
B. spielmanii | PSigHH | 0.1–10,000 |
B. turcica | 10,000 | |
B. valaisiana | VS116 | 0.1–10,000 |
Primer/Probe | Name | Channel | Sequence |
---|---|---|---|
Babesia sense | Piro18S-238s | 3 | 5′-TCGGTGATTCATAATAAACTRGCGAATCGC-3′ |
Babesia antisense | Piro18S-380as | 5′-GAATCGAACCCCAATTCCCCGTTACCCG-3′ | |
Babesia probe | Piro18S-340 | 5′-Cy5- GACGGTAGGGTATTGGCCTACCG-BHQ2-3′ | |
Bartonella sense | BsppITS325s | 1 | 5’-CCTCAGATGATGATCCCAAGCCTTCTGGCG-3’ |
Bartonella antisense | BsppITS543as | 5′-TAAAYTGGTGGGCCTGGGAGGACTTG-3′ | |
Bartonella probe | BsppITS500 | 5′-FAM-GTTAGAGCGCGCGCTTGATAAG -IABkFQ-3′ | |
Borrelia sense | BobuITS120s | 2 | 5′-AGGTCATTTTGGGGGTTTAGCTCAGTTGGCT-3′ |
Borrelia antisense | BoLymeITS200as | 5′-AATGGAGGTTAAGGGACTCGAACCCT-3′ | |
Borrelia probe | BobuITS160 | 5′-HEX-CGGCTTTGCAAGCCGAGGGTCAAG-BHQ-2-3′ |
Babesia Species | Theileria-Cytauxzoon | |
---|---|---|
B. bicornis | B. microti | T. cervi |
B. bigemina | B. negevi-like | T. equi |
B. c. canis | B. vulpes (former B. annae) | C. felis |
B. c. vogeli | maned Wolf Babesia | |
B. coco | Bsp AJB 2006 * | |
B. conradae | Bsp woodrat * | |
B. divergens | Bspp skunks (SK04 *, SK08 *) | |
B. duncani | Bspp raccoons (R214 *, 9879 *, CO8 *) | |
B. gibsoni | Bspp bears (NC08 *, NC13 *) | |
B. lengau |
Bartonella Species | |||
---|---|---|---|
B. henselae | B. alsatica | B. doshiae | B. rochalimae |
B. koehlerae | B. melophagi | B. schoembuchensis | B. tamiae |
B. quintana | B. volans | B. rattimassiliensis | Ca. B. k. boulousii |
B. v. berkhoffii TI | B. elizabethae | B. tribocorum | Ca. B. k. bothierii |
B. v. berkhoffii TII | B. monaki | B. chomelii | Bsp deer (D2 *, D4 *, D5 *, D6 *) |
B. v. berkhoffii TIII | B.v. baker | B. washoensis | Bsp river otter * |
B. clarridgeiae | B. bovis | B. vinsonii | Bsp mongoose * |
Borrelia Species (Strains) | |
---|---|
B. afzelii (Pko) | B. lusitanae (PotB2) |
B. bavariensis (PBi) | B. miyamotoi |
B. bissettii (PGeb) | B. spielmanii (PSigII) |
B. burgdorferi (B31, PBre) | B. turcica |
B. coriaceae | B. turicatae |
B. garinii (PBr, PHei, Pla, PRef, PWudII) | B. valaisiana (VS116) |
B. hermsii |
Borrelia sp. | Strain | Detection Limit (Genome Copies per 5 µL Sample) |
---|---|---|
B. afzelii | Pko | 1–10 |
B. bavariensis | VS116 | 0.1–10 |
B. bissettii | Pgeb | 1–10 |
B. burgdorferi | B31, Pbre | 1–10 |
B. garinii | PBr, Phei, Pla, Pref, PWudII | 1–10 |
B. hermsii | NA | |
B. lusitanae | PotiB2 | 1–10 |
B. miyamotoi | NA | |
B. spielmanii | PSigHH | 1–10 |
B. turcica | 1–10 | |
B. valaisiana | VS116 | 10 |
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Maggi, R.; Breitschwerdt, E.B.; Qurollo, B.; Miller, J.C. Development of a Multiplex Droplet Digital PCR Assay for the Detection of Babesia, Bartonella, and Borrelia Species. Pathogens 2021, 10, 1462. https://doi.org/10.3390/pathogens10111462
Maggi R, Breitschwerdt EB, Qurollo B, Miller JC. Development of a Multiplex Droplet Digital PCR Assay for the Detection of Babesia, Bartonella, and Borrelia Species. Pathogens. 2021; 10(11):1462. https://doi.org/10.3390/pathogens10111462
Chicago/Turabian StyleMaggi, Ricardo, Edward B. Breitschwerdt, Barbara Qurollo, and Jennifer C. Miller. 2021. "Development of a Multiplex Droplet Digital PCR Assay for the Detection of Babesia, Bartonella, and Borrelia Species" Pathogens 10, no. 11: 1462. https://doi.org/10.3390/pathogens10111462