Detection of Anaplasma Phagocytophilum in Horses With Suspected Tick-Borne Disease in Northeastern United States by Metagenomic Sequencing.
Abstract: Metagenomic sequencing of clinical diagnostic specimens has a potential for unbiased detection of infectious agents, diagnosis of polymicrobial infections and discovery of emerging pathogens. Herein, next generation sequencing (NGS)-based metagenomic approach was used to investigate the cause of illness in a subset of horses recruited for a tick-borne disease surveillance study during 2017-2019. Blood samples collected from 10 horses with suspected tick-borne infection and five apparently healthy horses were subjected to metagenomic analysis. Total genomic DNA extracted from the blood samples were enriched for microbial DNA and subjected to shotgun next generation sequencing using Nextera DNA Flex library preparation kit and V2 chemistry sequencing kit on the Illumina MiSeq sequencing platform. Overall, 0.4-0.6 million reads per sample were analyzed using Kraken metagenomic sequence classification program. The taxonomic classification of the reads indicated that bacterial genomes were overrepresented (0.5 to 1%) among the total microbial reads. Most of the bacterial reads (~91%) belonged to phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria and Tenericutes in both groups. Importantly, 10-42.5% of Alphaproteobacterial reads in 5 of 10 animals with suspected tick-borne infection were identified as Anaplasma phagocytophilum. Of the 5 animals positive for A. phagocytophilum sequence reads, four animals tested A. phagocytophilum positive by PCR. Two animals with suspected tick-borne infection and A. phagocytophilum positive by PCR were found negative for any tick-borne microbial reads by metagenomic analysis. The present study demonstrates the usefulness of the NGS-based metagenomic analysis approach for the detection of blood-borne microbes.
Copyright © 2021 Subbiah, Thirumalapura, Thompson, Kuchipudi, Jayarao and Tewari.
Publication Date: 2021-06-09 PubMed ID: 34179165PubMed Central: PMC8219919DOI: 10.3389/fvets.2021.673193Google Scholar: Lookup
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- Journal Article
Summary
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This research explores the use of next-generation sequencing (NGS)-based metagenomic analysis to identify infectious agents in horses with suspected tick-borne infections in the northeastern United States. Results revealed that this method allows for the detection of blood-borne microbes and could help improve the diagnosis of tick-borne diseases.
Methodology and Results
- The study used NGS-based metagenomic analysis to determine the cause of illness in horses believed to be suffering from tick-borne diseases between 2017 and 2019.
- Blood samples were collected from 10 horses with suspected tick-borne infection and five apparently healthy horses.
- The genomic DNA extracted from the samples was enriched for microbial DNA and then subjected to shotgun next-generation sequencing.
- Bacterial genomes were overrepresented among the total microbial reads.
- Importantly, it was discovered that in 5 out of 10 animals with a suspected tick-borne infection, 10-42.5% of Alphaproteobacterial reads were identified as Anaplasma Phagocytophilum.
- Of these 5 animals with Anaplasma Phagocytophilum sequence reads, four tested positive for Anaplasma Phagocytophilum via PCR (polymerase chain reaction), a method used to amplify small segments of DNA.
- Interestingly, two horses with suspected tick-borne infection that tested positive for Anaplasma Phagocytophilum via PCR were found to show no sign of any tick-borne microbial reads by the metagenomic analysis.
Conclusion and Implications
- This research demonstrates the effectiveness of using NGS-based metagenomic analysis for the detection of blood-borne microbes in horses potentially infected by tick-borne diseases.
- However, the results also indicate that PCR testing and metagenomic analysis may not always provide consistent results.
- This suggests that the NGS-based metagenomic analysis may be a valuable tool for initial screening and identification, but may need to be supplemented with PCR for final confirmation.
- Overall, the use of metagenomics for diagnosing tick-borne diseases could potentially lead to improvements in detection and treatment practices.
Cite This Article
APA
Subbiah M, Thirumalapura N, Thompson D, Kuchipudi SV, Jayarao B, Tewari D.
(2021).
Detection of Anaplasma Phagocytophilum in Horses With Suspected Tick-Borne Disease in Northeastern United States by Metagenomic Sequencing.
Front Vet Sci, 8, 673193.
https://doi.org/10.3389/fvets.2021.673193 Publication
Researcher Affiliations
- Pennsylvania Veterinary Laboratory, Harrisburg, PA, United States.
- Pennsylvania Veterinary Laboratory, Harrisburg, PA, United States.
- Pennsylvania Veterinary Laboratory, Harrisburg, PA, United States.
- Animal Diagnostic Laboratory, Pennsylvania State University, University Park, PA, United States.
- Center for Infectious Disease Dynamics, Pennsylvania State University, University Park, PA, United States.
- Animal Diagnostic Laboratory, Pennsylvania State University, University Park, PA, United States.
- Center for Infectious Disease Dynamics, Pennsylvania State University, University Park, PA, United States.
- Pennsylvania Veterinary Laboratory, Harrisburg, PA, United States.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Citations
This article has been cited 3 times.- Mauri Pablo JD, Del Solar JJC, Hinojosa Enciso ET, Polveiro RC, Vieira DDS, Ramos Sanchez EM, Bardales Escalante W, Maicelo Quintana JL, Lopez Lapa RM. Anaplasmosis in the Amazon: diagnostic challenges, persistence, and control of Anaplasma marginale and Anaplasma phagocytophilum. Front Vet Sci 2025;12:1571694.
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