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Volume 31, Number 6—June 2025

Research Letter

Francisella tularensis Subspecies holarctica in Stranded Beluga Whales, Cook Inlet, Alaska, USA

Natalie RouseComments to Author , Jeremy Buttler, Kristy Pabilonia, Christina Weller, Laurel Respicio-Kingry, Elizabeth Dietrich, Jeannine Petersen, Ganna Kovalenko, Eric Bortz, and Kathy Burek Huntington
Author affiliation: Alaska Veterinary Pathology Services, Eagle River, Alaska, USA (N. Rouse, K. Burek Huntington); University of Alaska Anchorage, Anchorage, Alaska, USA (N. Rouse, J. Buttler, G. Kovalenko, E. Bortz); Colorado State University, Fort Collins, Colorado, USA (K. Pabilonia, C. Weller); Centers for Disease Control and Prevention, Fort Collins (L. Respicio-Kingry, E. Dietrich, J. Petersen)

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Table

Tests performed on dead stranded beluga whales infected with Francisella tularensis, Cook Inlet, Alaska, USA*

Procedure Method Method detail Laboratory Manufacturer† or reference
Gross necropsy
 NA
 Tissues stored in whirl paks and swabs in cryovials with VTM or TSB with 15% glycerol and frozen at −80°C within 6 hours of sampling.
 AVPS
 Remel (VTM), Hardy Diagnostics (TSB)
Nanopore metagenomics
 QiAMP DNA/RNA kit
 Eluted in 50 μL elution buffer
 UAA
 QIAGEN
Nanopore metagenomics
 Rapid SMART9N
 Superscript IV TR-ase (Thermo Fisher Scientific†); primers RLB RT-9N, TTTTTCGTGCGCCGCTTCAACNNNNNNNNN and RLB TSO-RNA (r-hybrid oligo), GCTAATCATTGCTTTTTCGTGCGCCGCTTCAACATrGrGrG without DNase treatment. Cycling conditions: 42°C for 90 min; 70°C for 10 min; 4°C hold.
 UAA
 ‡
Nanopore metagenomics
 ONT† SQK-RPB114.96 kit (V14)
 LongAmp Taq (2X; New England BioLabs†) PCR (95°C for 45 min, then 30 cycles at 95°C for 15 s; 56°C for 15 s; 65°C for 5 min; and 10 min final extension) with Rapid PCR barcoding using ONT SQK-RPB114.96 kit V14 on an ONT MinION Mk1B device running MinKNOW v.24.06.8 with high-accuracy basecalling.
 UAA
 (4)
Nanopore metagenomics: bioinformatics sequence read classification
 wf-metagenomics and wf-alignment in Epi2me Labs v.5.1.3
 NA
 UAA
 Epi2me Labs
Nanopore metagenomics: F. tularensis genome mapping
 Reference-based assembly
 Minimap2
 UAA
 NA
Nanopore metagenomics: sequence allignment and annotation
 tbCon and ggplot in RStudio v. 2024.04.0+735
 NA
 UAA
 NA
MLST: amplification
 Amplification of 6 genes: fabH, tpiA, sdhA, rpoA, groEL, and pgm
 Primers described in references
 CDC
 (5–7)
MLST: sequencing of amplicon libraries
 Multiplexed amplicon sequencing on MiSeq platform
 Nextera XT and V2 300 cycle reagent kit
 CDC
 Illumina
MLST: F. tularensis genome mapping
 Reads were mapped to reference genes from F. tularensis subsp. holarctica LVS
 CLC Genomics Workbench
 CDC
 QIAGEN
MLST: phylogenetic tree construction
 Maximum-likelihood analysis
 Generalized time-reversible nucleotide substitution model with gamma distribution (4 categories) plus invariant sites and 1,000 bootstrap replications
 CDC
 MEGAX
F. tularensis culture
 LRN protocol
 NA
 CDC
 CDC LRN
F. tularensis PCR
 LRN protocol
 NA
 CSU VDL
 CDC LRN
Erysipelothrix sp. PCR
 NA
 Primers ERy4423F and Ery4587R only
 AVDL
 (8)
Influenza A virus PCR
 NA
 Primers described in reference
 Tufts and UAA
 (9)
Histopathology
 Hematoxylin and eosin staining
 NA
 HCS and AVPS
 NA
Immunohistochemistry for F. tularensis
 NA
 NA
 KSU
 NA
Saxitoxin
 ELISA
 NA
 WARRN West
 Abraxis
Domoic acid ELISA NA WARRN West Abraxis

*AVDL, University of Georgia Athens Veterinary Diagnostic Laboratory; AVPS, Alaska Veterinary Pathology Services; CDC, Centers for Disease Control and Prevention; CSU VDL, Colorado State University Veterinary Diagnostic Laboratory; HCS, Histological Consulting Services; KSU, Kansas State University Veterinary Diagnostic Laboratory; LRN, CDC Laboratory Response Network; LVS, live vaccine strain; MLST, multilocus sequence typing; NA, not applicable; ONT, Oxford Nanopore Technologies; TSB, tryptic soy broth; Tufts, Tufts University; UAA, University of Alaska; VTM, viral transport medium; WARRN West, National Oceanic and Atmospheric Administration Wildlife Algal-toxin Research and Response Network West. †Manufacturers and products: Abraxis, https://www.bms.com; Epi2me Labs, https://epi2me.nanoporetech.com; Hardy Diagnostics, https://hardydiagnostics.com; MEGAX, https://www.megasoftware.net; Minimap2, https://github.com/lh3/minimap2; New England Biolabs, https://www.neb.com; Oxford Nanopore Technologies, https://nanoporetech.com; QIAGEN, https://www.qiagen.com; Remel, https://www.thermofisher.com; RStudio, https://posit.co; tbCon, https://github.com/jeremyButtler/bioTools; Thermo Fisher Scientific; https://www.thermofisher.com. ‡I.M. Claro et al., unpub. data, https://doi.org/10.12688/wellcomeopenres.17170.2. §GenBank accession no. NC_007880.1.

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