http://vdi.sagepub.com/Investigation

Journal of Veterinary Diagnostic

http://vdi.sagepub.com/content/22/5/784The online version of this article can be found at:

 DOI: 10.1177/104063871002200525

2010 22: 784J VET Diagn InvestMulrooney, Robin Weiss, Judith Galeota and Annette Bredthauer

Sabrina L. Swenson, Leo G. Koster, Melinda Jenkins-Moore, Mary L. Killian, Emilio E. DeBess, Rocky J. Baker, Donna in Pet FerretsInfluenza A VirusNatural Cases of 2009 Pandemic H1N1

  

Published by:

http://www.sagepublications.com

On behalf of: 

  Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.

can be found at:Journal of Veterinary Diagnostic InvestigationAdditional services and information for    

  http://vdi.sagepub.com/cgi/alertsEmail Alerts:

 

http://vdi.sagepub.com/subscriptionsSubscriptions:  

http://www.sagepub.com/journalsReprints.navReprints:  

http://www.sagepub.com/journalsPermissions.navPermissions:  

What is This? 

- Sep 1, 2010Version of Record >>

by guest on October 11, 2013vdi.sagepub.comDownloaded from by guest on October 11, 2013vdi.sagepub.comDownloaded from by guest on October 11, 2013vdi.sagepub.comDownloaded from by guest on October 11, 2013vdi.sagepub.comDownloaded from by guest on October 11, 2013vdi.sagepub.comDownloaded from by guest on October 11, 2013vdi.sagepub.comDownloaded from by guest on October 11, 2013vdi.sagepub.comDownloaded from

J Vet Diagn Invest 22:784–788 (2010)

Natural cases of 2009 pandemic H1N1 Influenza A virus in pet ferrets

Sabrina L. Swenson,1 Leo G. Koster, Melinda Jenkins-Moore, Mary L. Killian, Emilio E. DeBess,Rocky J. Baker, Donna Mulrooney, Robin Weiss, Judith Galeota, Annette Bredthauer

Abstract. Respiratory swab samples were collected from 5 pet ferrets (Mustela putorius furo) exhibitinginfluenza-like illness. The ferrets represented 3 households in 2 states. In each case, the owners reportedinfluenza-like illness in themselves or family members prior to the onset of a similar illness in the ferrets. Real-time reverse transcription polymerase chain reaction assays designed for the detection of the 2009 H1N1Influenza A virus were conducted in the state animal health laboratories. The assays included detection of thematrix gene of Influenza A virus and neuraminidase gene specific for 2009 H1N1 virus. Samples were positivefor both screening assays. The samples were confirmed positive by the National Veterinary ServicesLaboratories. The history of illness in family members prior to illness in the ferrets suggests that Influenza Avirus was transmitted from humans to the ferrets.

Key words: Ferrets; Influenza A virus; transmission.

<!?show "fnote_aff1"$^!"content-markup(./author-grp[1]/aff|./author-grp[1]/dept-list)>During October 2009, 5 pet ferrets (Mustela putorius

furo) with influenza-like illness (ILI) representing 3households in 2 states (Oregon and Nebraska) weresampled and tested for the 2009 H1N1 Influenza A virusthat was first reported in humans in April 2009.2 Case 1was a 4-year-old ferret exhibiting sneezing, coughing,inappetence, nasal discharge, fever of 40.2uC, and generaldepression. The owner reported having ILI that started6 days prior to the ferret’s exhibiting clinical signs.Respiratory discharge was collected from the ferret usinga polyester swab by the referring veterinarian and wasplaced in saline for transport to the Oregon State

From the Diagnostic Virology Laboratory, National VeterinaryServices Laboratories, U.S. Department of Agriculture, Animal andPlant Health Inspection Service, Ames, IA (Swenson, Koster, Jenkins-Moore, Killian), Oregon Department of Human Services, Acute andCommunicable Disease Section, Portland, OR (DeBess), OregonState University, Veterinary Diagnostic Laboratory, Corvallis, OR(Baker, Mulrooney, Weiss), University of Nebraska, VeterinaryDiagnostic Center, Lincoln, NE (Galeota), and Nebraska Depart-ment of Health and Human Services, Lincoln, NE (Bredthauer).

1 Corresponding Author: Sabrina L. Swenson, National VeterinaryServices Laboratories, PO Box 844, 1920 Dayton Avenue, Ames, IA50010. sabrina.l.swenson@aphis.usda.gov

784 Case Reports

University Veterinary Diagnostic Laboratory (OSUVDL;Corvallis, Oregon) for testing. The ferret was treated withsubcutaneous fluids and antibiotics and was force fed bysyringe for 3 days. The ferret exhibited general weaknessthroughout the recovery period and slept unless awakenedby the owner. The ferret recovered without complications.The OSUVDL is a member of the National Animal HealthLaboratory Network and conducted real-time reversetranscription polymerase chain reaction (real-time RT-PCR) assays for the 2009 H1N1 virus matrix (M) andneuraminidase (NA) genes using the standardized protocolprovided by the National Veterinary Services Laboratories(NVSL). The matrix assay is based on an assay developed fordetection of Avian influenza virus.10 The modified matrixassay includes an additional reverse primer that is sequencespecific for the 2009 H1N1 matrix nucleic acid.11 The NAreal-time RT-PCR is a differential assay that specificallydetects the N1 viral RNA of the 2009 H1N1 virus.11 Both theM and NA real-time RT-PCR assays were positive withthreshold cycle (Ct) values of 29.2 and 31.0, respectively.

The original specimen was forwarded to NVSL (Ames,Iowa) for confirmatory testing, where the sample wasconfirmed by real-time RT-PCR to be positive for both theM and NA (N1) genes. An aliquot of the original specimenwas cultured on Madin–Darby canine kidney (MDCK)cells per NVSL protocol. Briefly, cell culture flasks ofMDCK cells at 70–90% confluency were rinsed with cellculture medium containing trypsin. Inoculum was added,and the flask was incubated at 37uC for 50–90 min. At theend of the incubation period, the flask was rinsed with cellculture medium, and replacement medium containingtrypsin was added. The flask was incubated at 37uC untilcytopathic effects were observed. The flask was frozen at270uC and thawed, and virus was harvested for furthertesting. The virus isolate was positive on both the M andNA real-time RT-PCR assays. The entire gene sequence ofthe hemagglutinin (HA), M, and NA genes was acquiredbidirectionally with 100% gene coverage. Briefly, RNA wasextracted with a commercial kita in accordance withmanufacturer’s instructions. Individual influenza viral

Figure 1. Phylogenetic relationship of the hemagglutinin gene of 2 pandemic 2009 H1N1 Influenza A virus isolates from ferrets inOregon (A/ferret/OR/23775/2009, A/ferret/OR/27004/2009) with other phylogenetic lineages of H1 influenza virus.

Case Reports 785

genes were amplified by RT-PCR as previously described.12

The amplicons were purified directly or from agarose gelsusing commercial kits.b The purified PCR products weresequenced at the NVSL using an automated system.a

Sequences were aligned with the ClustalW method.Phylogenetic and molecular evolutionary analyses wereconducted using MEGA version 4 using the neighbor-

joining tree-building method,13 with 1,000 bootstrapreplicates. Analysis of the HA, NA, and M genes wasdone with genes from viruses that were phylogeneticallyrepresentative of other lineages and included genes fromviruses from multiple sources (wild birds, poultry, andmammals) and from multiple geographical regions includ-ing North America, Europe, and Asia (National Center for

Figure 2. Phylogenetic relationship of the matrix gene of 2 pandemic 2009 H1N1 Influenza A virus isolates from ferrets in Oregon(A/ferret/OR/23775/2009, A/ferret/OR/27004/2009) with other phylogenetic lineages of influenza virus.

786 Case Reports

Biotechnology Information Influenza Virus Resource,http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html).Phylogenetic analyses of the 3 gene segments indicated thatthe virus was most closely related to the 2009 H1N1 viruscompared with other swine, avian, and human viruses(Figs. 1–3; GU953235, GU953236, GU953237).

Case 2 involved a household of 9 ferrets. Familymembers of the household reported ILI approximately1 week before the ferrets became ill. Affected familymembers reportedly did not have direct contact with theferrets. The household also contained 2 cats and 1 dog. NoILI was noted in these companion animals. Three of theferrets aged 5–6 years were examined by a veterinarian.Two ferrets exhibited sneezing, coughing, nasal discharge,and fevers .39.4uC. The third ferret exhibited a fever of39.3uC. The remaining 6 ferrets became ill over a 2–3 daytime period after the first ferret exhibited clinical signs. All9 ferrets recovered uneventfully from the ILI. Nasal swabswere collected from the 3 ferrets examined by the referringveterinarian and placed in saline for transport to theOSUVDL. Samples were tested as noted above with all 3

swabs identified as positive on the matrix (Ct values of 22.9,26.5, and 23.1) and NA (Ct values of 25.9, 29.5, and 26.0)assays. The original specimens were forwarded to NVSLfor confirmatory testing. All 3 clinical specimens wereconfirmed positive on the M and NA real-time RT-PCRassays and were inoculated on MDCK cells. Virus wasisolated from all 3 clinical specimens. Two of the 3 isolatesobtained underwent HA, M, and NA sequencing and wereconfirmed to be the 2009 H1N1 virus (Figs. 1–3; GU332633,GU332634, GU332635; not shown GU979208, GU979209,GU979210).

Case 3 involved a household of 4 ferrets. Three childrenand the father reported sequential symptoms of ILI (ferretsrepeatedly exposed as family members became ill) begin-ning approximately 9 days prior to ILI in the ferrets. TheILI in the children included coughing, nasal discharge,chills, and fever .39.4uC. The father also experienced bodyaches, diarrhea, and difficult breathing. The father visited aphysician and tested positive on a rapid Influenza A virustest 4 days prior to the ferrets becoming ill. Theasymptomatic mother was the primary caretaker of the

Figure 3. Phylogenetic relationship of the neuraminidase gene of 2 pandemic 2009 H1N1 Influenza A virus isolates from ferrets inOregon (A/ferret/OR/23775/2009, A/ferret/OR/27004/2009) with other phylogenetic lineages of N1 influenza virus.

Case Reports 787

ferrets while the family members were ill, but it was notedthat the children cuddled the ferrets during this time period.The ages of the 4 ferrets were 1 year (n 5 2), 6 years, and8 years. All 4 ferrets exhibited bilateral nasal discharge withcoughing and/or sneezing. The 8-year-old neutered maleexperienced severe symptoms and was presented to aveterinarian for examination and treatment 4 days afterthe ILI first appeared in the ferrets. This ferret hadextensive bilateral nasal discharge, was dehydrated, andhad a low normal temperature (37.8uC). The ferret wastreated with subcutaneous fluids, antibiotics, and support-ive care but died 6 days after being presented to theveterinarian for treatment. A nasal swab collected by theveterinarian was submitted to the University of NebraskaVeterinary Diagnostic Center (Lincoln, Nebraska) and testedas reported above. Both the M and NA assays were positivewith Ct values of 36.9 and 36.3, respectively. The originalspecimen was submitted to NVSL and confirmed positive onthe M and NA real-time RT-PCR assays. Virus was notisolated from the original clinical specimen, and directsequencing from the clinical specimen was unsuccessful.

While it cannot be conclusively proven that people werethe source of influenza for the ferrets, the ILI in familymembers prior to ILI in the ferrets and a positive InfluenzaA virus rapid test in 1 family member of case 3 prior toclinical symptoms in the family ferrets are stronglysuggestive that people were the source of infection in eachcase. The fact that ferrets are known to become infectedwith human influenza viruses and are routinely used asmodels in human influenza studies supports a conclusion ofhumans as a source of influenza for ferrets.5,6 Ferretsexperimentally infected with the 2009 H1N1 virus demon-strated clinical signs compatible with what was reported inthe ferrets from the 3 households.7 The 3 gene segmentssequenced from each of the 2009 H1N1 viruses examinedfrom the ferrets were clustered in the same region as theMexico, California, and New York 2009 H1N1 humanisolates that were obtained early in the pandemic influenzaoutbreak in the spring of 2009 and were distinct from apreviously reported naturally occurring influenza infectionof ferrets.8 To the authors’ knowledge, these are the firstreports of natural infection of pet ferrets with the 2009H1N1 virus and also the first reports of probabletransmission from humans to ferrets. Transmission of the2009 H1N1 virus also has been observed between peopleand a pet cat.11 The movement of influenza from onespecies to another is not unusual although not demonstratedpreviously for the 2009 H1N1 virus until the recent reportin the cat and the ferrets in the current study. Equineinfluenza virus subtype H3N8 has been found to infectdogs, and both cats and dogs have been infected withAvian influenza virus subtype H5N1.1,3,4,9,14 The potentialfor the 2009 H1N1 Influenza A virus to move betweenspecies, especially companion animals with close humancontacts, demonstrates the need for strong interactionsbetween animal health and human health.

Acknowledgements. The authors thank the followingveterinarians for submission of samples and histories to thestate diagnostic laboratories: Drs. Ross Weinstein (Ore-gon), Christy Cutting (Oregon), and Tammy Craig(Nebraska). The authors also wish to thank the followingtechnicians for diagnostic support in the labs: AnnetteOlson (Iowa), Marlys Rodamaker (Iowa), Stephanie Lex(Iowa), and Julia Russ (Nebraska).

Sources and manufacturers

a. MagMAXTM AI/ND Viral RNA Isolation Kit, 3130xl Genetic

Analyzer Applied Biosystems, Austin, TX.

b. QIAquickH PCR Purification Kit, QIAquickH Gel Extraction

Kit, Qiagen Inc., Valencia, CA.

References

1. Crawford PC, Dubovi EJ, Castleman WL, et al.: 2005,

Transmission of equine influenza virus to dogs. Science

310:482–485.

2. Garten RJ, Davis CT, Russell CA, et al.: 2009, Antigenic and

genetic characterization of swine-origin 2009 A(H1N1)

influenza viruses circulating in humans. Science 325:197–201.

3. Harder TC, Vahlenkamp TW: 2010, Influenza virus infections

in dogs and cats. Vet Immunol Immunopathol 134:54–60.

4. Kuiken T, Rimmelzwaan G, van Riel D, et al.: 2004, Avian

H5N1 influenza in cats. Science 306:241.

5. Maher JA, Destefano J: 2004, The ferret: an animal model to

study influenza virus. Lab Anim 33:50–53.

6. Matsuoka Y, Lamirande EW, Subbarao K: 2009, The ferret

model for influenza. Curr Protoc Microbiol 13:15G.2.1–

15G.2.29.

7. Munster VJ, de Wit E, van den Brand JMA, et al.: 2009,

Pathogenesis and transmission of swine-origin 2009 A(H1N1)

influenza virus in ferrets. Science 325:481–483.

8. Patterson AR, Cooper VL, Yoon KJ, et al.: 2009, Naturally

occurring influenza infection in a ferret (Mustela putorius furo)

colony. J Vet Diagn Invest 21:527–530.

9. Payungporn S, Crawford PC, Kouo TS, et al.: 2008, Influenza

A virus (H3N8) in dogs with respiratory disease, Florida.

Emerg Infect Dis 14:902–908.

10. Spackman E, Senne DA, Myers TJ, et al.: 2002, Development

of a real-time reverse transcriptase assay for type A influenza

virus and the avian H5 and H7 hemagglutinin subtypes. J Clin

Microbiol 40:3256–3260.

11. Sponseller B, Strait E, Jergens A, et al.: 2010, Influenza A

pandemic (H1N1) 2009 virus infection in domestic cat. Emerg

Infect Dis 16:534–537.

12. Suarez DL, Garcia M, Latimer J, et al.: 1999, Phylogenetic

analysis of H7 avian influenza viruses isolated from the live

bird markets of the Northeast United States. J Virol

73:3567–3573.

13. Tamura K, Dudley J, Nei M, Kumar S: 2007, MEGA4:

Molecular Evolutionary Genetics Analysis (MEGA) software

version 4.0. Mol Biol Evol 24:1596–1599.

14. Thiry E, Zicola A, Addie D, et al.: 2007, Highly pathogenic

avian influenza H5N1 virus in cats and other carnivores. Vet

Microbiol 122:25–31.

788 Case Reports

Journal of Veterinary Diagnostic Investigation24(4) 813© 2012 The Author(s)Reprints and permission: sagepub.com/journalsPermissions.navDOI: 10.1177/1040638712450578http://jvdi.sagepub.com

Erratum

450578 JVDXXX10.1177/1040638712450578

Corrigendum

Stegelmeier, BL, et al.: 2010, Experimental rayless goldenrod (Isocoma pluriflora) toxicosis in goats. J Vet Diagn Invest. 22: 570–577

In the article “Experimental rayless goldenrod (Isocoma pluriflora) toxicosis in goats” by Bryan L. Stegelmeier et al., the published mean body weight and the means and statistics of serum biochemistries were carried out on groups of 4 animals, not 3, as described in the Material and Methods section. The additional animal in each group was part of an auxiliary physi-ologic study and though the animals were dosed and treated the same, they were not necropsied and were not included in the histologic study. To correct this oversight, the corrected weight and chemistry table (shaded cells indicate corrected numbers) are listed below. The differences are minimal and do not alter the conclusions. In addition, reference 7 has been deleted.

Material and Methods: “Fifteen, yearling, female Spanish goats weighing 29.4 ± 3.4 kg (mean ± standard deviation) were randomly divided into 5 groups with 3 animals per group.”

References: Reference 7 should be deleted

Corrected Table 1. Selected mean serum biochemical data from groups of 3 goats dosed with rayless goldenrod (Isocoma pluriflora) to obtain benzofuran ketone doses of 0, 10, 20, 40, and 60 mg/kg body weight for 7 days.*

Serum result (mean ± standard deviation)

Serum test (reference range†) Dose Day 0 Day 3 Day 6 Day 7

Creatinine kinase (< 350 U/l) 0 226 ± 93 107 ± 6 73 ± 16a 66 ± 30a

10 226 ± 160 118 ± 8 206 ± 184a 495 ± 623ab

20 967 ± 1233 306 ± 276 240 ± 113a 497 ± 277ab

40 125 ± 18 117 ± 24 6,699 ± 5,329b 16,270 ± 11,054b

60 202 ± 93 202 ± 124 2,987 ± 3,701a 10,433 ± 4,326ab

Cardiac troponin-I (<0.40 U/l‡) 0 <0.02 ± 0.0 <0.02 ± 0.0 <0.02 ± 0.0 <0.02 ± 0.0 10 <0.02 ± 0.0 <0.02 ± 0.0 <0.02 ± 0.0 <0.02 ± 0.0 20 <0.02 ± 0.0 0.17 ± 0.26 0.05 ± 0.03 <0.02 ± 0.0 40 <0.02 ± 0.0 <0.02 ± 0.0 1.98 ± 3.39 1.79 ± 2.97 60 <0.02 ± 0.0 <0.02 ± 0.0 1.38 ± 2.31 0.13 ± 0.18Aspartate aminotransferase (<125 U/l) 0 96 ± 7 91 ± 6 83 ± 2a 72 ± 3a

10 147 ± 69 104 ± 11 89 ± 8a 97 ± 13a

20 164 ± 82 284 ± 248 293 ± 252ab 376 ± 256a

40 112 ± 17 102 ± 12 991 ± 184c 3,277 ± 1,556b

60 96 ± 13 115 ± 31 819 ± 571bc 2,095 ± 1,333b

Alanine aminotransferase (<55 U/l) 0 39 ± 3 37 ± 3 38 ± 0a 43 ± 18a

10 44 ± 1 42 ± 3 39 ± 2a 37 ± 1a

20 41 ± 9 57 ± 34 63 ± 38ab 61 ± 25a

40 46 ± 2 44 ± 4 134 ± 24a 333 ± 127b

60 40 ± 7 44 ± 5 118 ± 84ab 267 ± 176b

Lactate dehydrogenase (<1,560 U/l) 0 1,061 ± 145 1,075 ± 62 875 ± 213a 573 ± 115a

10 1,334 ± 668 1,050 ± 223 942 ± 265a 709 ± 182a

20 1,650 ± 1,546 2,617 ± 2,685 1,185 ± 449a 753 ± 447a

40 1,054 ± 201 1,162 ± 130 5,996 ± 2,491b 9,891 ± 3,210b

60 1,026 ± 287 1,277 ± 348 3,623 ± 2,924ab 7,011 ± 5,205a

*Different means (<0.05) between groups are indicated with superscript letters.†Estimates of normal range were determined as 2 standard deviations from mean values of control goats and pretreatment samples. These ranges are probably laboratory and assay specific.‡Cardiac troponin-I concentrations below detection limits are reported as <0.02 ng/ml.