In August 2021, a large-scale mortality event affected harbor porpoises (Phocoena phocoena) in the Netherlands. Pathology and ancillary testing of 22 animals indicated that the most likely cause of death was Erysipelothrix rhusiopathiae infection. This zoonotic agent poses a health hazard for cetaceans and possibly for persons handling cetacean carcasses

Begeman, Lineke

Broens, Els M

der Graaf-van Bloois, Linda van

Duim, Birgitta

Gröne, Andrea

IJsseldijk, Lonneke L

Kik, Marja J L

Klijnstra, Mirjam D

Lakemeyer, Jan

Leopold, Mardik F

Munnink, Bas B Oude

Ten Doeschate, Mariel

van Schalkwijk, Linde

Zomer, Aldert

Utrecht University Repository

patient’s consent was not obtained. The patient has not had a recurrence of multiple ulcers but remains positive for H. ailurogastricus. The limitation of this case report is that, although we succeeded in culturing H. ailurogas-tricus in the stomach of this patient and the drug-sus-ceptibility test has determined the regimen for eradica-tion, we have not yet been able to perform eradication therapy. Therefore, the efficacy of eradication in H. ailu-rogastricus infections has not been confirmed. H. ailuro-gastricus eradication therapy will be administered at the next patient visit to prevent ulcer recurrence. The clinical importance of NHPH infection in the human stomach has been increasing in the post–H. pylori era. Because NHPH species such as H. suis and H. ailurogastricus cannot be detected by most H. pylori diag-nostic tests, such as the urea breath test and stool anti-gen test, NHPH infections should be considered when routine H. pylori tests are negative, despite the presence of inflammatory findings in the gastric mucosa.This work was supported by MEXT/JSPS KAKENHI (grant no. 20H03667, awarded to H.S.), and by AMED (grant no. JP20fk0108148, awarded to E.R. and H.S.).About the AuthorDr. Sano is an instructor at the Division of Gastroenterology and Hepatology, Tokai University School of Medicine. His primary research interests are endoscopic treatment of gastrointestinal diseases and elucidation of pathological mechanisms.References  1. Nishizawa T, Suzuki H. Mechanisms of Helicobacter pylori antibiotic resistance and molecular testing. Front Mol Biosci. 2014;1:19. https://doi.org/10.3389/fmolb.2014.00019  2. Rimbara E, Suzuki M, Matsui H, Nakamura M, Morimoto M, Sasakawa C, et al. Isolation and characterization of Helicobacter suis from human stomach. Proc Natl Acad Sci U S A. 2021;118:e2026337118. https://doi.org/10.1073/pnas.2026337118  3. Nakamura M, Øverby A, Michimae H, Matsui H,  Takahashi S, Mabe K, et al. PCR analysis and specific  immunohistochemistry revealing a high prevalence of  non–Helicobacter pylori Helicobacters in Helicobacter  pylori–negative gastric disease patients in Japan: high susceptibility to an Hp eradication regimen. Helicobacter. 2020;25:e12700. https://doi.org/10.1111/hel.12700  4. Shafaie S, Kaboosi H, Peyravii Ghadikolaii F. Prevalence  of non Helicobacter pylori gastric Helicobacters in Iranian dyspeptic patients. BMC Gastroenterol. 2020;20:190.  https://doi.org/10.1186/s12876-020-01331-x  5. Joosten M, Lindén S, Rossi M, Tay AC, Skoog E, Padra M,  et al. Divergence between the highly virulent zoonotic  pathogen Helicobacter heilmannii and its closest relative, the low-virulence “Helicobacter ailurogastricus” sp. nov. Infect  Immun. 2015;84:293–306. https://doi.org/10.1128/IAI.01300-15Address for correspondence: Hidekazu Suzuki, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan; email: hsuzuki@tokai.ac.jpHarbor Porpoise Deaths  Associated with Erysipelothrix rhusiopathiae, the  Netherlands, 2021Lonneke L. IJsseldijk, Lineke Begeman,  Birgitta Duim, Andrea Gröne, Marja J.L. Kik,  Mirjam D. Klijnstra, Jan Lakemeyer,  Mardik F. Leopold, Bas B. Oude Munnink,  Mariel ten Doeschate, Linde van Schalkwijk,  Aldert Zomer, Linda van der Graaf-van Bloois,  Els M. BroensAuthor affiliations: Utrecht University, Utrecht, the Netherlands (L.L. IJsseldijk, B. Duim, A. Gröne, M.J.L. Kik, J. Lakemeyer,  M. ten Doeschate, L. van Schalkwijk, A. Zomer, L. van der  Graaf-van Bloois, E.M. Broens); Erasmus University Medical Center, Rotterdam, the Netherlands (L. Begeman, B.B. Oude Munnink); Wageningen Food Safety Research, Wageningen, the Netherlands (M.D. Klijnstra); Wageningen Marine Research,  Den Helder, the Netherlands (M.F. Leopold)DOI: https://doi.org/10.3201/eid2903.221698Erysipelothrix bacteria cause infections in humans and other species after contact with infected ani-mals or environmental sources (1). Illness ranges from mild to systemic, which can include septicemia and endocarditis. Erysipelothrix can survive for long peri-ods in the environment, including marine ecosystems (1) associated with marine fish, mollusks, and crus-taceans. Erysipelothrix infection affects captive and  Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 4, April 2023 835RESEARCH LETTERSIn August 2021, a large-scale mortality event affected har-bor porpoises (Phocoena phocoena) in the Netherlands. Pathology and ancillary testing of 22 animals indicated that the most likely cause of death was Erysipelothrix rhu-siopathiae infection. This zoonotic agent poses a health hazard for cetaceans and possibly for persons handling cetacean carcasses.free-ranging crustaceans and is linked to fatal sepsis (2). To our knowledge, reports of large-scale mortal-ity events caused by Erysipelothrix infection in marine mammals are absent from the literature, and Erysip-elothrix has not been detected in stranded porpoises along the Netherlands coastline since the start of our harbor porpoise stranding research program in 2008.At the end of August 2021, a total of 190 dead harbor porpoises (Phocoena phocoena) were found on Dutch Wadden islands; the annual average for stranded harbor porpoises on the entire Dutch coast-line is 600. No anthropogenic activities in the south-ern or central North Sea that could explain this mor-tality event were reported to the government of the Netherlands in the 4–6 weeks before the event.Most porpoises were found in an advanced state of decomposition. Twenty-two animals were collected for examination at the Faculty of Veterinary Medicine of Utrecht University (Appendix Table 1, https://wwwnc.cdc.gov/EID/article/29/4/22-1698-App1.pdf). We immediately necropsied 2, and the rest were temporarily frozen pending postmortem investiga-tion and ancillary testing.Because of advanced decomposition, we could perform only gross pathologic examinations and sam-pling for ancillary testing, following a standardized international protocol (3). Adult female porpoises were mostly in good to moderate nutritional con-dition with mild to moderate parasitic infections of various organs and had been reproductively active (Appendix Table 1). Of the 21 stomachs examined (1 was not examined because of gross damage caused by scavengers), none contained marine debris; 10 con-tained the remains of a few prey, reflecting nonrecent food intake, and the remaining stomachs were empty.Samples from 3 porpoises with gross changes (mammary gland, lung, spinal cord) were cultured on blood agar (bioTrading, https://biotrading.com) at 37°C for 48 h. Culture results were positive for Ery-sipelothrix rhusiopathiae. Subsequently, we tested liver samples from 21 animals for E. rhusiopathiae; and 16 were positive (Appendix Table 2). To investigate the relatedness of isolates, genomes of 18 isolates were se-quenced by using Illumina NextSeq (https://www.il-lumina.com) and assembled by using SPAdes version 3.14.1 (4); we included 11 publicly available reference genomes from different E. rhusiopathiae clades (5). A core genome alignment was made with Parsnp ver-son 1.2 (6) and visualized by using iTol version 4 (7).Genomes from this study were phylogenetically positioned between clade 2 E. rhusiopathiae reference genomes and formed 2 distinct clusters showing ≈3,400 single-nucleotide polymorphism (SNP) differences and limited diversity of <6 SNPs within the clusters (Figure). That pattern suggests dissemination of 2 clon-al lineages of E. rhusiopathiae, either through exposure to a common source or contact between individuals.Virology tests on 14 fecal, 15 blood, and 17 spleen samples and metagenomic sequencing with VirCapSeq-VERT (8) revealed no virus sequences of 836 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 4, April 2023RESEARCH LETTERSFigure. Phylogenetic tree of Erysipelothrix rhusiopathiae from stranded harbor porpoises, the Netherlands, 2021, compared with reference genomes described by Forde et al. (5). Branches are square root transformed. Detailed information for each sample is provided in Appendix Table 3 (https://wwwnc.cdc.gov/EID/article/29/4/22-1698-App1.pdf).interest. In addition, we tested 20 lung and 20 brain samples for influenza A virus, paramyxoviruses (in-cluding morbilliviruses), coronaviruses (including SARS-CoV-2), and herpesviruses. Only 2 brain sam-ples tested positive for P. phocoena alphaherpesvirus (Appendix Table 1), described as an incidental cause of death in porpoises (9). Our results indicate that vi-ruses were an unlikely factor in this mortality event.We pooled 20 stomach content samples and 21 liver samples in triplicate and analyzed them with a Liquid Chromatograph Triple Quadrupole Mass Spectrometer (LC-MS/MS) (McCrone Associates, https://www.mccrone.com) for domoic acid, saxi-toxins, tetrodotoxin, and lipophilic marine toxins. Only saxitoxin was detected; it was in 1 pooled liver sample (estimated concentration 15 μg/kg). Subse-quently, we analyzed livers individually, and saxi-toxin was not confirmed in any of the individual sam-ples. We therefore conclude that harmful algae were an unlikely factor in this mortality event.Gross pathologic assessment revealed a moderate to good body condition for most porpoises, but none had recently fed. This finding suggests a subacute cause of death from sudden and excessive disease. No clinically relevant viruses were detected. Phycotoxins were detected in a limited number of porpoises. In contrast, E. rhusiopathiae was isolated from most in-vestigated porpoises. Therefore, we consider E. rhusio-pathiae to be the most likely cause of death. Advanced autolysis of the carcasses made detection of distinc-tive lesions associated with Erysipelothrix infection im-possible. The low number of SNPs differing between isolates suggests common exposure, possibly a food source, transmission between porpoises, or both.Our results draw attention to possibly increased cetacean susceptibility to E. rhusiopathiae, to new or emerging sources of Erysipelothrix in the marine en-vironment, or both. Erysipelothrix remains viable in a carcass up to 12 days in direct sunlight, up to 4 months in putrefied flesh, and up to 9 months in a buried car-cass (10). This new emerging source and the long sur-vival time in carcasses demonstrates a need for hav-ing only trained personnel handle stranded animals, proper disposal of carcasses, and increased aware-ness for the potential presence and transmission of this zoonotic bacterium among cetaceans.AcknowledgmentsWe thank all volunteers and organizations of the Dutch Stranding Network for their tremendous efforts and work to document stranded porpoises during the event.  Necropsies were assisted by Louis van den Boom,  Natashja Ennen-Buijs, Olle Juch, Darryl Leydekkers, Immelie Coenen Morales, Jan Mosterd, Eva Schotanus, and Ruby Wagensveld. We also thank Judith van den Brand for her input in the discussions of the ancillary testing. Virology was conducted at Erasmus MC, with special thanks to Marco van de Bildt, Irina Chestakova, Marjan Boter, Babette Weller, Reina Sikkema, and Marion Koopmans. Bacteriology was conducted at the Veterinary Microbiological Diagnostic Center, for which we thank all staff involved. Stomach investigations were conducted at Wageningen Marine Research with the help of Guido Keijl and Eva Schotanus. Algae toxin research was carried out by Wageningen Food Safety Research with the help of Domenique van der Horst.The research was financed by the Ministry of Agriculture, Nature and Food Quality (project no. 1400012118), for which we are especially grateful for the help of  Anne-Marie Svoboda and Sandra van der Graaf.About the AuthorDr. IJsseldijk is assistant professor at the Division of Pathology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University.  Her primary research interests focus on marine mammal health and conservation.References  1. Wang Q, Chang BJ, Riley TV. Erysipelothrix rhusiopathiae. Vet Microbiol. 2010;140:405–17.  https://doi.org/10.1016/ j.vetmic.2009.08.012  2. Ceccolini ME, Wessels M, Macgregor SK, Deaville R,  Perkins M, Jepson PD, et al. Systemic Erysipelothrix  rhusiopathiae in seven free-ranging delphinids stranded in England and Wales. Dis Aquat Organ. 2021;145:173–84.  https://doi.org/10.3354/dao03609  3. IJsseldijk LL, Brownlow AC, Mazzariol S, editors. Best practice for cetacean post mortem investigation and tissue sampling. Joint ACCOBAMS and ASCOBANS document. Stralsund, Germany: Agreement for the Conservation of  Cetaceans of the Baltic Sea, Mediterranean Sea and  Contiguous Atlantic Area (ACCOBAMS); Agreement on the Conservation of Small Cetaceans of the Baltic, North East Atlantic, Irish and North Seas (ASCOBANS); 2019 [cited 2022 Nov 7]. https://www.ascobans.org/sites/default/files/ document/ascobans_ac25_inf3.2_rev1_best-practice- cetacean-post-mortem-investigation.pdf  4. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly  algorithm and its applications to single-cell sequencing.  J Comput Biol. 2012;19:455–77.  https://doi.org/10.1089/cmb.2012.0021  5. Forde T, Biek R, Zadoks R, Workentine ML, De Buck J, Kutz S, et al. Genomic analysis of the multi-host pathogen  Erysipelothrix rhusiopathiae reveals extensive recombination  as well as the existence of three generalist clades with wide  geographic distribution. BMC Genomics. 2016;17:461.  https://doi.org/10.1186/s12864-016-2643-0  6. Treangen TJ, Ondov BD, Koren S, Phillippy AM. The Harvest suite for rapid core-genome alignment and visualization of  Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 4, April 2023 837RESEARCH LETTERSthousands of intraspecific microbial genomes. Genome Biol. 2014;15:524. https://doi.org/10.1186/s13059-014-0524-x  7. Letunic I, Bork P. Interactive Tree Of Life (iTOL) v4:  recent updates and new developments. Nucleic Acids Res. 2019;47(W1):W256–9. https://doi.org/10.1093/nar/gkz239  8. Briese T, Kapoor A, Mishra N, Jain K, Kumar A, Jabado OJ, et al. Virome capture sequencing enables sensitive viral diagnosis and comprehensive virome analysis. MBio. 2015;6:e01491–15. https://doi.org/10.1128/mBio.01491-15  9. van Elk C, van de Bildt M, van Run P, de Jong A, Getu S, Verjans G, et al. Central nervous system disease and genital disease in harbor porpoises (Phocoena phocoena) are  associated with different herpesviruses. Vet Res (Faisalabad). 2016;47:28.  https://doi.org/10.1186/s13567-016-0310-8  10. Brooke CJ, Riley TV. Erysipelothrix rhusiopathiae: bacteriology, epidemiology and clinical manifestations of an occupational pathogen. J Med Microbiol. 1999;48:789–99. https://doi.org/ 10.1099/00222615-48-9-789Address for correspondence: Els M. Broens, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 TD, Utrecht, the Netherlands; email: e.m.broens@uu.nlPowassan Virus Infection  Detected by Metagenomic Next-Generation Sequencing, Ohio, USAMarjorie Farrington,1 Jamie Elenz, Matthew Ginsberg, Charles Y. Chiu, Steve Miller, Scott F. PangonisAuthor affiliations: Children’s Hospital Medical Center of Akron,  Akron, Ohio, USA (M. Farrington, M. Ginsberg, S.F Pangonis); Columbiana County Health District, Lisbon, Ohio, USA (J. Elenz); University of California, San Francisco, California, USA (C.Y. Chiu, S. Miller)DOI: https://doi.org/10.3201/eid2904.221005Powassan virus (POWV) is a tickborne flavivirus that causes encephalitis with severe neurologic sequelae (1). In the United States, POWV infections occur primarily in the Northeast and Great Lakes re-gions (2). We report a case of human POWV infection in Ohio.A 4-year-old boy was brought to the emergency department because of fever, vomiting, and convul-sive status epilepticus. He had no neurologic history or developmental delays. Mosquito and tick exposure history was substantial, although no engorged ticks were recently removed. The patient had not traveled outside of Ohio. Results of a computed tomography scan of the head were unremarkable. We initiated intravenous vancomycin, ceftriaxone, and acyclovir. Magnetic resonance imaging showed left temporal pulvinar and thalamic T2-weighted fluid attenuated inver-sion recovery hyperintensity and restricted diffu-sion; an electroencephalogram showed lateralized periodic discharges. Cerebrospinal fluid (CSF) was collected by lumbar puncture, revealing a leu-kocyte count of 44 cells/μL (reference range <10 cells/μL) of which 85% were lymphocytes; glucose and protein levels were normal. The patient’s BIO-FIRE FILMARRAY Meningitis/Encephalitis PCR panel (bioMérieux, https://www.biomerieux-di-agnostics.com) was negative (Table). He was admit-ted to the pediatric intensive care unit, and seizures were controlled with anticonvulsants. Tests for infectious and noninfectious causes of meningitis and encephalitis were negative (Table). Antimicro-bial drugs were discontinued after negative bacte-rial cultures were observed. Acyclovir was discon-tinued after PCR of CSF for herpes simplex virus was negative. On hospitalization day 5, severe neurologic de-cline developed, and brain magnetic resonance imag-ing was repeated. New areas of T2 hyperintensity and restricted diffusion and thalamic microhemorrhages in a rhombencephalitis pattern were identified. Lum-bar puncture was repeated, revealing considerable lymphocytic pleocytosis and elevated protein (156 mg/dL). Leading diagnoses were autoimmune en-cephalitis and acute necrotizing encephalopathy of childhood (ANEC). The patient exhibited severe en-cephalopathy, nystagmus, right hemiparesis, and diffuse hypertonia. He was treated with high dose methylprednisolone, plasmapheresis, and intrave-nous immunoglobulins. Genetic testing for familial ANEC type 1 was nega-tive. We sent CSF obtained on hospital day 5 to the Uni-versity of California San Francisco for metagenomic 838 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 4, April 2023RESEARCH LETTERS1Current affiliation: St. Louis Children’s Hospital, St. Louis, Missouri, USA.We describe a 4-year-old male patient in Ohio, USA, who had encephalitis caused by Powassan virus lineage 2. Vi-rus was detected by using metagenomic next-generation sequencing and confirmed with IgM and plaque reduction neutralization assays. Clinicians should recognize chang-ing epidemiology of tickborne viruses to enhance enceph-alitis diagnosis and management.

Harbor Porpoise Deaths Associated with Erysipelothrix rhusiopathiae, the Netherlands, 2021

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Harbor Porpoise Deaths Associated with Erysipelothrix rhusiopathiae, the Netherlands, 2021

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