Abstract Background Hemorrhagic pneumonia is a disease of farmed mink (<it>Neovison vison</it>) caused by <it>Pseudomonas aeruginosa</it>. The disease is highly seasonal in Danish mink with outbreaks occurring almost exclusively in the autumn. Human respiratory syncytial virus (RSV) has been shown to augment infection with <it>P. aeruginosa</it> in mice and to promote adhesion of <it>P. aeruginosa</it> to human respiratory cells. Findings We tested 50 lung specimens from mink with hemorrhagic pneumonia for bovine RSV by reverse transcriptase polymerase chain reaction (PCR) and for human RSV by a commercial real-time PCR. RSV was not found. Conclusions This study indicates that human and bovine RSV is not a major co-factor for development of hemorrhagic pneumonia in Danish mink.</p

AJ Easton

AM de Vrankrijker

Anne S Hammer

B Knox

BE Van Ewijk

Centers for Disease Control and Prevention (CDC)

Charlotte M Salomonsen

E Honda

GA Prince

GG Long

HK Johansen

Jeanette Jakobsen

JM Hament

JM Stark

JY Homma

K Nordstoga

Lars E Larsen

LE Larsen

LG Byrd

LR Yeolekar

Niels Høiby

SolvejØ Breum

V Avadhanula

PubMed

Abstract Background Hemorrhagic pneumonia is a disease of farmed mink (Neovison vison) caused by Pseudomonas aeruginosa. The disease is highly seasonal in Danish mink with outbreaks occurring almost exclusively in the autumn. Human respiratory syncytial virus (RSV) has been shown to augment infection with P. aeruginosa in mice and to promote adhesion of P. aeruginosa to human respiratory cells. Findings We tested 50 lung specimens from mink with hemorrhagic pneumonia for bovine RSV by reverse transcriptase polymerase chain reaction (PCR) and for human RSV by a commercial real-time PCR. RSV was not found. Conclusions This study indicates that human and bovine RSV is not a major co-factor for development of hemorrhagic pneumonia in Danish mink.</p

Salomonsen Charlotte M

Breum SolvejØ

Larsen Lars E

Jakobsen Jeanette

Høiby Niels

Hammer Anne S

Directory of Open Access Journals

Acta Veterinaria Scandinavica

Investigation of the presence of human or bovine respiratory syncytial virus in the lungs of mink (<it>Neovison vison</it>) with hemorrhagic pneumonia due to <it>Pseudomonas aeruginosa</it>

Salomonsen, Charlotte Mark

Breum, Solvej Østergaard

Larsen, Lars Erik

Jakobsen, Jeanette

Høiby, Niels

Hammer, Anne Sofie Vedsted

Copenhagen University Research Information System

u n i ve r s i t y  o f  co pe n h ag e n  Københavns UniversitetInvestigation of the presence of human or bovine respiratory syncytial virus in thelungs of mink (Neovison vison) with hemorrhagic pneumonia due to PseudomonasaeruginosaSalomonsen, Charlotte Mark; Breum, Solvej Østergaard; Larsen, Lars Erik; Jakobsen,Jeanette; Høiby, Niels; Hammer, Anne Sofie VedstedPublished in:Acta Veterinaria Scandinavica (Online)DOI:10.1186/1751-0147-54-70Publication date:2012Document versionPublisher's PDF, also known as Version of recordCitation for published version (APA):Salomonsen, C. M., Breum, S. Ø., Larsen, L. E., Jakobsen, J., Høiby, N., & Hammer, A. S. V. (2012).Investigation of the presence of human or bovine respiratory syncytial virus in the lungs of mink (Neovison vison)with hemorrhagic pneumonia due to Pseudomonas aeruginosa. Acta Veterinaria Scandinavica (Online), 54(70).https://doi.org/10.1186/1751-0147-54-70Download date: 02. Feb. 2020Salomonsen et al. Acta Veterinaria Scandinavica 2012, 54:70http://www.actavetscand.com/content/54/1/70BRIEF COMMUNICATION Open AccessInvestigation of the presence of human or bovinerespiratory syncytial virus in the lungs of mink(Neovison vison) with hemorrhagic pneumoniadue to Pseudomonas aeruginosaCharlotte M Salomonsen1*, Solvej Ø Breum1, Lars E Larsen1, Jeanette Jakobsen2, Niels Høiby2and Anne S Hammer1,3AbstractBackground: Hemorrhagic pneumonia is a disease of farmed mink (Neovison vison) caused by Pseudomonasaeruginosa. The disease is highly seasonal in Danish mink with outbreaks occurring almost exclusively in theautumn. Human respiratory syncytial virus (RSV) has been shown to augment infection with P. aeruginosa in miceand to promote adhesion of P. aeruginosa to human respiratory cells.Findings: We tested 50 lung specimens from mink with hemorrhagic pneumonia for bovine RSV by reversetranscriptase polymerase chain reaction (PCR) and for human RSV by a commercial real-time PCR. RSV was notfound.Conclusions: This study indicates that human and bovine RSV is not a major co-factor for development ofhemorrhagic pneumonia in Danish mink.Keywords: Hemorrhagic pneumonia, Mink, Pseudomonas aeruginosa, Respiratory syncytial virusFindingsPseudomonas aeruginosa was identified as the cause ofhemorrhagic pneumonia in farmed mink (Neovisonvison) in 1953 [1]. This disease can cause mortalities ashigh as 50% on the farm level [2,3] but can also follow amilder course [1]. Hemorrhagic pneumonia is character-ized by a rapid disease progression in the individualmink and it is highly seasonal in Denmark with farmoutbreaks occurring almost exclusively from Septemberto November. Whether the seasonal pattern is related tothe seasonal life cycle of farmed mink or if it relates toco-infection with other pathogens has not been investi-gated. Thus, no infectious co-factors have been identi-fied but the highly seasonal appearance of disease mayindicate the presence of a viral co-factor. In human cys-tic fibrosis (CF) patients, a seasonal pattern for acute* Correspondence: charlottemarksoerensen@hotmail.com1National Veterinary Institute, Technical University of Denmark, Bülowsvej 27,DK-1870, Frederiksberg C, DenmarkFull list of author information is available at the end of the article© 2012 Salomonsen et al.; licensee BioMed CeCreative Commons Attribution License (http:/distribution, and reproduction in any mediumand chronic P. aeruginosa infection has been identified[4] and the authors proposed respiratory viruses as theunderlying cause for this pattern. Respiratory viruseshave a seasonal infection pattern coinciding with that ofP. aeruginosa infection in CF patients and respiratoryviruses are known to precede bacterial infections [5,6].Respiratory syncytial virus (RSV) is an envelopedsingle-stranded negative sense RNA virus belonging tothe genus Pneumovirus in the family Paramyxoviridae.Variants of the virus infect various species includingsheep, goats, mice, cattle and humans. A number of ani-mal species can be experimentally infected with humanRSV (HRSV) [7,8]. The occurrence of HRSV infectionsin humans is known to be highly seasonal with the ma-jority of cases occurring in the winter or spring [9].HRSV has been shown to promote adhesion of P. aeru-ginosa to cells in the respiratory tract of mice [10,11]and to directly mediate binding of P. aeruginosa tohuman epithelial cells in vitro [12]. HRSV has also beenshown to replicate in mink lung cells in vitro [13] and inntral Ltd. This is an Open Access article distributed under the terms of the/creativecommons.org/licenses/by/2.0), which permits unrestricted use,, provided the original work is properly cited.Salomonsen et al. Acta Veterinaria Scandinavica 2012, 54:70 Page 2 of 3http://www.actavetscand.com/content/54/1/70the lungs of infant ferrets [14]. RSV has never beendescribed as naturally occurring in mink and experimen-tal studies on RSV infections in mink has not been pub-lished. However mink may be exposed to HRSV by theirhuman caretakers and to bovine RSV (BRSV) from thefeed which often include slaughter offal from cattle.The aim of the present study was to investigatewhether HRSV or BRSV could be found in lung tissuefrom mink succumbing to hemorrhagic pneumoniacaused by P. aeruginosa and hence possibly be part ofthe pathogenesis of this disease.Fifty samples of lung tissue from Danish mink wereselected of which 40 originated from mink submittedfrom 10 fur farms for routine diagnostic examinationand found to have hemorrhagic pneumonia associatedwith culturing of P. aeruginosa in pure culture from thelungs. Twenty of these mink originated from three farmswith high mortality due to hemorrhagic pneumonia(above 15%) while 15 mink were submitted from fivefarms with low mortality (below 2%). Data on mortalitywas not available for the last five mink submitted forroutine diagnostic investigation. Ten samples originatedfrom normal wildmink experimentally infected intra-nasally with 103-109 viable P. aeruginosa bacteria. Twoof the ten experimentally infected mink developed clin-ical symptoms of hemorrhagic pneumonia (lethargy,labored breathing) and were euthanized two days afterinoculation and necropsied immediately. The remainingeight clinically normal mink were euthanized eight daysafter inoculation and necropsied immediately. Theseeight mink served as negative controls. The animalexperiments were approved by the Danish AnimalExperiments Inspectorate. From the control mink, speci-mens of the left caudal lobe was obtained, while lung tis-sue with gross lesions of pneumonia was chosen fromthe experimentally infected clinically diseased mink andthe mink submitted for routine diagnostic. The sampleswere collected during 2011 and stored at −20°C untilfurther testing.Total RNA was purified from the lung tissue using theRNeasy Mini Kit (Qiagen, Düsseldorf, Germany) on aQIAcube (Qiagen) according to manufacturer’s instruc-tions. A BRSV positive cell culture was used for positivecontrol while nuclease-free water (Amresco, Solon, OH,USA) was used as a negative control of the purificationstep. The recovered RNA was stored at −80°C until fur-ther analysis.The presence of BRSV was tested with a modificationof a previously published conventional reverse tran-scriptase polymerase chain reaction (RT-PCR) assay tar-geting the F-gene with the forward primer sequence 50-AACCGGCTTCCTTCAGTAGAGC-30 and the reverseprimer sequence 50-CAATACCACCCACGATCTGTCC-30 [15]. The RT-PCR was performed on a Biometra T3Thermocycler (Biometra, Goettingen, Germany) in atotal volume of 25 μl using Qiagen OneStep RT-PCR kit(Qiagen) and 3 μl of extracted RNA, 400 μM dNTP mixand 500 nM of each primer. The amplificationtemperature profile was 30 min at 50°C for reverse tran-scription, 15 min at 95°C and 35 cycles of 30 s at 95°C,30 s at 55°C and 1 min at 72°C, followed by 10 min at72°C. In each PCR reaction a known BRSV positive sam-ple and a non-template control was run with the sam-ples as controls. PCR products with the expected size of730 bp were visualized on E-gelW 2% General PurposeAgarose (Invitrogen, Carlsbad, CA, USA).For detection of HRSV a commercially available real-time PCR (ProdesseW ProFlu™+, Gen-probe, San Diego,CA, USA) was employed. The primers used for this de-tection were not available due to the commercial natureof the PCR. The amplification and detection were per-formed on a MX3005p (Stratagene, La Jolla, CA, USA).The manufacturer’s protocol was followed in detail.The 50 mink lung samples were negative when testedin the BRSV and HRSV specific assays. In 5 samplestested with the BRSV specific primers, weak PCR pro-ducts of the predicted size were recognized but sequenceanalysis showed no resemblance to the F gene of BRSVor to other known genes for these products. Therefore,they probably represent unspecific annealing to minkspecific nucleic acids. No PCR products were obtainedusing the HRSV specific assay.Evidence of concurrent infection with HRSV andBRSV and P. aeruginosa in cases of mink hemorrhagicpneumonia was not found. Whether this was due tobreakdown of the virus, e.g. due to severe inflammation,collection of samples after clearance of the virus, or be-cause HRSV or BRSV simply was not present in the lungsamples remains uncertain.Histological examinations of lungs from mink suffer-ing from hemorrhagic pneumonia have not revealed syn-cytial cells [16,17] but due to the severe lung lesionsseen in this type of infection, syncytial cells may be diffi-cult to recognize or may be necrotic at the time of histo-logical examination. RSV can experimentally infect anumber of species [8] but is apparently quite host-specific in eliciting disease even though the various RSVsare closely related [7]. If a specific mink RSV is existingand is associated with hemorrhagic pneumonia in mink,the primers used for detection of BRSV and HRSV inthis study may not be able to detect this virus. To fur-ther elucidate whether a novel RSV-like virus is presentin mink, other primer-sets may be generated based onhighly conserved parts of genomes belonging to thegenus Pneumovirus. Electron microscopy, immunohisto-chemistry of lungs and nasal epithelium or de novo se-quencing of tissue from animals showing acuterespiratory symptoms may also be a valuable tool forSalomonsen et al. Acta Veterinaria Scandinavica 2012, 54:70 Page 3 of 3http://www.actavetscand.com/content/54/1/70identifying unrecognized respiratory viruses that may actas co-factors to P. aeruginosa hemorrhagic pneumonia.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsCMS participated in the design of the study, identified and collected suitablematerial, coordinated the study and drafted the manuscript. SØB participatedin the design of the study, supervised the BRSV testing, interpreted thesequencing of the weak PCR products and revised the manuscript. LELcontributed to the study design and revised the manuscript. JJ planned theHRSV testing and performed the practical experiment. NH contributed to thestudy design. ASH participated in the design of the study and the initialidentification of tissue used in the study. All authors read and approved thefinal manuscript.AcknowledgementsThe authors wish to thank Kirstine Vorborg for excellent technical assistance.Nordvacc and Kopenhagen Fur are thanked for financial support of thisstudy. The funding sources were not involved in study design, datacollection, analysis, interpretation, writing or decision to publish the workpresented in this communication.Author details1National Veterinary Institute, Technical University of Denmark, Bülowsvej 27,DK-1870, Frederiksberg C, Denmark. 2Department of Clinical Microbiology,Rigshospitalet, Blegdamsvej 9, DK-2100, Copenhagen Ø, Denmark.3Department of Veterinary Disease Biology, Faculty of Health and MedicalSciences, University of Copenhagen, Ridebanevej 3, DK-1870, Frederiksberg C,Denmark.Received: 30 July 2012 Accepted: 19 November 2012Published: 26 November 2012References1. Knox B: Pseudomonas aeruginosa as a cause of enzootic infections inmink [in Danish]. Nord Vet Med 1953, 5:731.2. Honda E, Homma JY, Abe C, Tanamoto K, Noda H, Yanagawa R: Effects ofthe common protective antigen (OEP) and toxoids of protease andelastase from Pseudomonas aeruginosa on protection againsthemorrhagic pneumonia in mink. Zentralbl Bakteriol [Orig A] 1977,237:297–309.3. Homma JY, Abe C, Tanamoto K, Hirao Y, Morihara K, Tsuzuki H, Yanagawa R,Honda E, Aoi Y, Fujimoto Y, Goryo M, Imazeki N, Noda H, Goda A, TakeuchiS, Ishihara T: Effectiveness of immunization with single and multi-component vaccines prepared from a common antigen (OEP), proteaseand elastase toxoids of Pseudomonas aeruginosa on protection againsthemorrhagic pneumonia in mink due to P. aeruginosa. Jpn J Exp Med1978, 48:111–133.4. Johansen HK, Hoiby N: Seasonal onset of initial colonisation and chronicinfection with Pseudomonas aeruginosa in patients with cystic fibrosis inDenmark. Thorax 1992, 47:109–111.5. Hament JM, Kimpen JL, Fleer A, Wolfs TF: Respiratory viral infectionpredisposing for bacterial disease: a concise review. FEMS Immunol MedMicrobiol 1999, 26:189–195.6. Avadhanula V, Rodriguez CA, Devincenzo JP, Wang Y, Webby RJ, Ulett GC,Adderson EE: Respiratory viruses augment the adhesion of bacterialpathogens to respiratory epithelium in a viral species- and cell type-dependent manner. J Virol 2006, 80:1629–1636.7. Easton AJ, Domachowske JB, Rosenberg HF: Animal pneumoviruses:molecular genetics and pathogenesis. Clin Microbiol Rev 2004, 17:390–412.8. Byrd LG, Prince GA: Animal models of respiratory syncytial virus infection.Clin Infect Dis 1997, 25:1363–1368.9. Centers for Disease Control and Prevention (CDC): Brief report: respiratorysyncytial virus activity - United States, 2005–2006. MMWR Morb MortalWkly Rep 2006, 55:1277–1279.10. Stark JM, Stark MA, Colasurdo GN, LeVine AM: Decreased bacterialclearance from the lungs of mice following primary respiratory syncytialvirus infection. J Med Virol 2006, 78:829–838.11. de Vrankrijker AM, Wolfs TF, Ciofu O, Hoiby N, van der Ent CK, Poulsen SS,Johansen HK: Respiratory syncytial virus infection facilitates acutecolonization of Pseudomonas aeruginosa in mice. J Med Virol 2009,81:2096–2103.12. Van Ewijk BE, Wolfs TF, Aerts PC, Van Kessel KP, Fleer A, Kimpen JL, Van derEnt CK: RSV mediates Pseudomonas aeruginosa binding to cystic fibrosisand normal epithelial cells. Pediatr Res 2007, 61:398–403.13. Yeolekar LR, Damle RG, Basu A, Rao BL: Growth of respiratory syncytialvirus in mink lung epithelial cells. Indian J Med Res 2002, 116:255–257.14. Prince GA, Porter DD: The pathogenesis of respiratory syncytial virusinfection in infant ferrets. Am J Pathol 1976, 82:339–352.15. Larsen LE, Tjornehoj K, Viuff B, Jensen NE, Uttenthal A: Diagnosis ofenzootic pneumonia in Danish cattle: reverse transcription-polymerasechain reaction assay for detection of bovine respiratory syncytial virus innaturally and experimentally infected cattle. J Vet Diagn Invest 1999,11:416–422.16. Nordstoga K: Pseudomonas infections in mink with special reference topseudomonas vasculitis in pulmonary lesions. Acta Vet Scand 1968, 9:33–40.17. Long GG, Gallina AM, Gorham JR: Pseudomonas pneumonia of mink:pathogenesis, vaccination, and serologic studies. Am J Vet Res 1980,41:1720–1725.doi:10.1186/1751-0147-54-70Cite this article as: Salomonsen et al.: Investigation of the presence ofhuman or bovine respiratory syncytial virus in the lungs of mink(Neovison vison) with hemorrhagic pneumonia due to Pseudomonasaeruginosa. Acta Veterinaria Scandinavica 2012 54:70.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color ﬁgure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submit

English

Investigation of the presence of human or bovine respiratory syncytial virus in the lungs of mink (Neovison vison) with hemorrhagic pneumonia due to Pseudomonas aeruginosa

Springer - Publisher Connector

Salomonsen et al. Acta Veterinaria Scandinavica 2012, 54:70
http://www.actavetscand.com/content/54/1/70BRIEF COMMUNICATION Open AccessInvestigation of the presence of human or bovine
respiratory syncytial virus in the lungs of mink
(Neovison vison) with hemorrhagic pneumonia
due to Pseudomonas aeruginosa
Charlotte M Salomonsen1*, Solvej Ø Breum1, Lars E Larsen1, Jeanette Jakobsen2, Niels Høiby2
and Anne S Hammer1,3Abstract
Background: Hemorrhagic pneumonia is a disease of farmed mink (Neovison vison) caused by Pseudomonas
aeruginosa. The disease is highly seasonal in Danish mink with outbreaks occurring almost exclusively in the
autumn. Human respiratory syncytial virus (RSV) has been shown to augment infection with P. aeruginosa in mice
and to promote adhesion of P. aeruginosa to human respiratory cells.
Findings: We tested 50 lung specimens from mink with hemorrhagic pneumonia for bovine RSV by reverse
transcriptase polymerase chain reaction (PCR) and for human RSV by a commercial real-time PCR. RSV was not
found.
Conclusions: This study indicates that human and bovine RSV is not a major co-factor for development of
hemorrhagic pneumonia in Danish mink.
Keywords: Hemorrhagic pneumonia, Mink, Pseudomonas aeruginosa, Respiratory syncytial virusFindings
Pseudomonas aeruginosa was identified as the cause of
hemorrhagic pneumonia in farmed mink (Neovison
vison) in 1953 [1]. This disease can cause mortalities as
high as 50% on the farm level [2,3] but can also follow a
milder course [1]. Hemorrhagic pneumonia is character-
ized by a rapid disease progression in the individual
mink and it is highly seasonal in Denmark with farm
outbreaks occurring almost exclusively from September
to November. Whether the seasonal pattern is related to
the seasonal life cycle of farmed mink or if it relates to
co-infection with other pathogens has not been investi-
gated. Thus, no infectious co-factors have been identi-
fied but the highly seasonal appearance of disease may
indicate the presence of a viral co-factor. In human cys-
tic fibrosis (CF) patients, a seasonal pattern for acute* Correspondence: charlottemarksoerensen@hotmail.com
1National Veterinary Institute, Technical University of Denmark, Bülowsvej 27,
DK-1870, Frederiksberg C, Denmark
Full list of author information is available at the end of the article
© 2012 Salomonsen et al.; licensee BioMed Ce
Creative Commons Attribution License (http:/
distribution, and reproduction in any mediumand chronic P. aeruginosa infection has been identified
[4] and the authors proposed respiratory viruses as the
underlying cause for this pattern. Respiratory viruses
have a seasonal infection pattern coinciding with that of
P. aeruginosa infection in CF patients and respiratory
viruses are known to precede bacterial infections [5,6].
Respiratory syncytial virus (RSV) is an enveloped
single-stranded negative sense RNA virus belonging to
the genus Pneumovirus in the family Paramyxoviridae.
Variants of the virus infect various species including
sheep, goats, mice, cattle and humans. A number of ani-
mal species can be experimentally infected with human
RSV (HRSV) [7,8]. The occurrence of HRSV infections
in humans is known to be highly seasonal with the ma-
jority of cases occurring in the winter or spring [9].
HRSV has been shown to promote adhesion of P. aeru-
ginosa to cells in the respiratory tract of mice [10,11]
and to directly mediate binding of P. aeruginosa to
human epithelial cells in vitro [12]. HRSV has also been
shown to replicate in mink lung cells in vitro [13] and inntral Ltd. This is an Open Access article distributed under the terms of the
/creativecommons.org/licenses/by/2.0), which permits unrestricted use,
, provided the original work is properly cited.
Salomonsen et al. Acta Veterinaria Scandinavica 2012, 54:70 Page 2 of 3
http://www.actavetscand.com/content/54/1/70the lungs of infant ferrets [14]. RSV has never been
described as naturally occurring in mink and experimen-
tal studies on RSV infections in mink has not been pub-
lished. However mink may be exposed to HRSV by their
human caretakers and to bovine RSV (BRSV) from the
feed which often include slaughter offal from cattle.
The aim of the present study was to investigate
whether HRSV or BRSV could be found in lung tissue
from mink succumbing to hemorrhagic pneumonia
caused by P. aeruginosa and hence possibly be part of
the pathogenesis of this disease.
Fifty samples of lung tissue from Danish mink were
selected of which 40 originated from mink submitted
from 10 fur farms for routine diagnostic examination
and found to have hemorrhagic pneumonia associated
with culturing of P. aeruginosa in pure culture from the
lungs. Twenty of these mink originated from three farms
with high mortality due to hemorrhagic pneumonia
(above 15%) while 15 mink were submitted from five
farms with low mortality (below 2%). Data on mortality
was not available for the last five mink submitted for
routine diagnostic investigation. Ten samples originated
from normal wildmink experimentally infected intra-
nasally with 103-109 viable P. aeruginosa bacteria. Two
of the ten experimentally infected mink developed clin-
ical symptoms of hemorrhagic pneumonia (lethargy,
labored breathing) and were euthanized two days after
inoculation and necropsied immediately. The remaining
eight clinically normal mink were euthanized eight days
after inoculation and necropsied immediately. These
eight mink served as negative controls. The animal
experiments were approved by the Danish Animal
Experiments Inspectorate. From the control mink, speci-
mens of the left caudal lobe was obtained, while lung tis-
sue with gross lesions of pneumonia was chosen from
the experimentally infected clinically diseased mink and
the mink submitted for routine diagnostic. The samples
were collected during 2011 and stored at −20°C until
further testing.
Total RNA was purified from the lung tissue using the
RNeasy Mini Kit (Qiagen, Düsseldorf, Germany) on a
QIAcube (Qiagen) according to manufacturer’s instruc-
tions. A BRSV positive cell culture was used for positive
control while nuclease-free water (Amresco, Solon, OH,
USA) was used as a negative control of the purification
step. The recovered RNA was stored at −80°C until fur-
ther analysis.
The presence of BRSV was tested with a modification
of a previously published conventional reverse tran-
scriptase polymerase chain reaction (RT-PCR) assay tar-
geting the F-gene with the forward primer sequence 50-
AACCGGCTTCCTTCAGTAGAGC-30 and the reverse
primer sequence 50-CAATACCACCCACGATCTGTCC-
30 [15]. The RT-PCR was performed on a Biometra T3Thermocycler (Biometra, Goettingen, Germany) in a
total volume of 25 μl using Qiagen OneStep RT-PCR kit
(Qiagen) and 3 μl of extracted RNA, 400 μM dNTP mix
and 500 nM of each primer. The amplification
temperature profile was 30 min at 50°C for reverse tran-
scription, 15 min at 95°C and 35 cycles of 30 s at 95°C,
30 s at 55°C and 1 min at 72°C, followed by 10 min at
72°C. In each PCR reaction a known BRSV positive sam-
ple and a non-template control was run with the sam-
ples as controls. PCR products with the expected size of
730 bp were visualized on E-gelW 2% General Purpose
Agarose (Invitrogen, Carlsbad, CA, USA).
For detection of HRSV a commercially available real-
time PCR (ProdesseW ProFlu™+, Gen-probe, San Diego,
CA, USA) was employed. The primers used for this de-
tection were not available due to the commercial nature
of the PCR. The amplification and detection were per-
formed on a MX3005p (Stratagene, La Jolla, CA, USA).
The manufacturer’s protocol was followed in detail.
The 50 mink lung samples were negative when tested
in the BRSV and HRSV specific assays. In 5 samples
tested with the BRSV specific primers, weak PCR pro-
ducts of the predicted size were recognized but sequence
analysis showed no resemblance to the F gene of BRSV
or to other known genes for these products. Therefore,
they probably represent unspecific annealing to mink
specific nucleic acids. No PCR products were obtained
using the HRSV specific assay.
Evidence of concurrent infection with HRSV and
BRSV and P. aeruginosa in cases of mink hemorrhagic
pneumonia was not found. Whether this was due to
breakdown of the virus, e.g. due to severe inflammation,
collection of samples after clearance of the virus, or be-
cause HRSV or BRSV simply was not present in the lung
samples remains uncertain.
Histological examinations of lungs from mink suffer-
ing from hemorrhagic pneumonia have not revealed syn-
cytial cells [16,17] but due to the severe lung lesions
seen in this type of infection, syncytial cells may be diffi-
cult to recognize or may be necrotic at the time of histo-
logical examination. RSV can experimentally infect a
number of species [8] but is apparently quite host-
specific in eliciting disease even though the various RSVs
are closely related [7]. If a specific mink RSV is existing
and is associated with hemorrhagic pneumonia in mink,
the primers used for detection of BRSV and HRSV in
this study may not be able to detect this virus. To fur-
ther elucidate whether a novel RSV-like virus is present
in mink, other primer-sets may be generated based on
highly conserved parts of genomes belonging to the
genus Pneumovirus. Electron microscopy, immunohisto-
chemistry of lungs and nasal epithelium or de novo se-
quencing of tissue from animals showing acute
respiratory symptoms may also be a valuable tool for
Salomonsen et al. Acta Veterinaria Scandinavica 2012, 54:70 Page 3 of 3
http://www.actavetscand.com/content/54/1/70identifying unrecognized respiratory viruses that may act
as co-factors to P. aeruginosa hemorrhagic pneumonia.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CMS participated in the design of the study, identified and collected suitable
material, coordinated the study and drafted the manuscript. SØB participated
in the design of the study, supervised the BRSV testing, interpreted the
sequencing of the weak PCR products and revised the manuscript. LEL
contributed to the study design and revised the manuscript. JJ planned the
HRSV testing and performed the practical experiment. NH contributed to the
study design. ASH participated in the design of the study and the initial
identification of tissue used in the study. All authors read and approved the
final manuscript.
Acknowledgements
The authors wish to thank Kirstine Vorborg for excellent technical assistance.
Nordvacc and Kopenhagen Fur are thanked for financial support of this
study. The funding sources were not involved in study design, data
collection, analysis, interpretation, writing or decision to publish the work
presented in this communication.
Author details
1National Veterinary Institute, Technical University of Denmark, Bülowsvej 27,
DK-1870, Frederiksberg C, Denmark. 2Department of Clinical Microbiology,
Rigshospitalet, Blegdamsvej 9, DK-2100, Copenhagen Ø, Denmark.
3Department of Veterinary Disease Biology, Faculty of Health and Medical
Sciences, University of Copenhagen, Ridebanevej 3, DK-1870, Frederiksberg C,
Denmark.
Received: 30 July 2012 Accepted: 19 November 2012
Published: 26 November 2012
References
1. Knox B: Pseudomonas aeruginosa as a cause of enzootic infections in
mink [in Danish]. Nord Vet Med 1953, 5:731.
2. Honda E, Homma JY, Abe C, Tanamoto K, Noda H, Yanagawa R: Effects of
the common protective antigen (OEP) and toxoids of protease and
elastase from Pseudomonas aeruginosa on protection against
hemorrhagic pneumonia in mink. Zentralbl Bakteriol [Orig A] 1977,
237:297–309.
3. Homma JY, Abe C, Tanamoto K, Hirao Y, Morihara K, Tsuzuki H, Yanagawa R,
Honda E, Aoi Y, Fujimoto Y, Goryo M, Imazeki N, Noda H, Goda A, Takeuchi
S, Ishihara T: Effectiveness of immunization with single and multi-
component vaccines prepared from a common antigen (OEP), protease
and elastase toxoids of Pseudomonas aeruginosa on protection against
hemorrhagic pneumonia in mink due to P. aeruginosa. Jpn J Exp Med
1978, 48:111–133.
4. Johansen HK, Hoiby N: Seasonal onset of initial colonisation and chronic
infection with Pseudomonas aeruginosa in patients with cystic fibrosis in
Denmark. Thorax 1992, 47:109–111.
5. Hament JM, Kimpen JL, Fleer A, Wolfs TF: Respiratory viral infection
predisposing for bacterial disease: a concise review. FEMS Immunol Med
Microbiol 1999, 26:189–195.
6. Avadhanula V, Rodriguez CA, Devincenzo JP, Wang Y, Webby RJ, Ulett GC,
Adderson EE: Respiratory viruses augment the adhesion of bacterial
pathogens to respiratory epithelium in a viral species- and cell type-
dependent manner. J Virol 2006, 80:1629–1636.
7. Easton AJ, Domachowske JB, Rosenberg HF: Animal pneumoviruses:
molecular genetics and pathogenesis. Clin Microbiol Rev 2004, 17:390–412.
8. Byrd LG, Prince GA: Animal models of respiratory syncytial virus infection.
Clin Infect Dis 1997, 25:1363–1368.
9. Centers for Disease Control and Prevention (CDC): Brief report: respiratory
syncytial virus activity - United States, 2005–2006. MMWR Morb Mortal
Wkly Rep 2006, 55:1277–1279.
10. Stark JM, Stark MA, Colasurdo GN, LeVine AM: Decreased bacterial
clearance from the lungs of mice following primary respiratory syncytial
virus infection. J Med Virol 2006, 78:829–838.11. de Vrankrijker AM, Wolfs TF, Ciofu O, Hoiby N, van der Ent CK, Poulsen SS,
Johansen HK: Respiratory syncytial virus infection facilitates acute
colonization of Pseudomonas aeruginosa in mice. J Med Virol 2009,
81:2096–2103.
12. Van Ewijk BE, Wolfs TF, Aerts PC, Van Kessel KP, Fleer A, Kimpen JL, Van der
Ent CK: RSV mediates Pseudomonas aeruginosa binding to cystic fibrosis
and normal epithelial cells. Pediatr Res 2007, 61:398–403.
13. Yeolekar LR, Damle RG, Basu A, Rao BL: Growth of respiratory syncytial
virus in mink lung epithelial cells. Indian J Med Res 2002, 116:255–257.
14. Prince GA, Porter DD: The pathogenesis of respiratory syncytial virus
infection in infant ferrets. Am J Pathol 1976, 82:339–352.
15. Larsen LE, Tjornehoj K, Viuff B, Jensen NE, Uttenthal A: Diagnosis of
enzootic pneumonia in Danish cattle: reverse transcription-polymerase
chain reaction assay for detection of bovine respiratory syncytial virus in
naturally and experimentally infected cattle. J Vet Diagn Invest 1999,
11:416–422.
16. Nordstoga K: Pseudomonas infections in mink with special reference to
pseudomonas vasculitis in pulmonary lesions. Acta Vet Scand 1968, 9:33–
40.
17. Long GG, Gallina AM, Gorham JR: Pseudomonas pneumonia of mink:
pathogenesis, vaccination, and serologic studies. Am J Vet Res 1980,
41:1720–1725.
doi:10.1186/1751-0147-54-70
Cite this article as: Salomonsen et al.: Investigation of the presence of
human or bovine respiratory syncytial virus in the lungs of mink
(Neovison vison) with hemorrhagic pneumonia due to Pseudomonas
aeruginosa. Acta Veterinaria Scandinavica 2012 54:70.Submit your next manuscript to BioMed Central
and take full advantage of: 
• Convenient online submission
• Thorough peer review
• No space constraints or color ﬁgure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at 
www.biomedcentral.com/submit


Investigation of the presence of human or bovine respiratory syncytial virus in the lungs of mink (Neovison vison) with hemorrhagic pneumonia due to Pseudomonas aeruginosa

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Salomonsen et al. Acta Veterinaria Scandinavica 2012, 54:70http://www.actavetscand.com/content/54/1/70BRIEF COMMUNICATION Open AccessInvestigation of the presence of human or bovinerespiratory syncytial virus in the lungs of mink(Neovison vison) with hemorrhagic pneumoniadue to Pseudomonas aeruginosaCharlotte M Salomonsen1*, Solvej Ø Breum1, Lars E Larsen1, Jeanette Jakobsen2, Niels Høiby2and Anne S Hammer1,3AbstractBackground: Hemorrhagic pneumonia is a disease of farmed mink (Neovison vison) caused by Pseudomonasaeruginosa. The disease is highly seasonal in Danish mink with outbreaks occurring almost exclusively in theautumn. Human respiratory syncytial virus (RSV) has been shown to augment infection with P. aeruginosa in miceand to promote adhesion of P. aeruginosa to human respiratory cells.Findings: We tested 50 lung specimens from mink with hemorrhagic pneumonia for bovine RSV by reversetranscriptase polymerase chain reaction (PCR) and for human RSV by a commercial real-time PCR. RSV was notfound.Conclusions: This study indicates that human and bovine RSV is not a major co-factor for development ofhemorrhagic pneumonia in Danish mink.Keywords: Hemorrhagic pneumonia, Mink, Pseudomonas aeruginosa, Respiratory syncytial virusFindingsPseudomonas aeruginosa was identified as the cause ofhemorrhagic pneumonia in farmed mink (Neovisonvison) in 1953 [1]. This disease can cause mortalities ashigh as 50% on the farm level [2,3] but can also follow amilder course [1]. Hemorrhagic pneumonia is character-ized by a rapid disease progression in the individualmink and it is highly seasonal in Denmark with farmoutbreaks occurring almost exclusively from Septemberto November. Whether the seasonal pattern is related tothe seasonal life cycle of farmed mink or if it relates toco-infection with other pathogens has not been investi-gated. Thus, no infectious co-factors have been identi-fied but the highly seasonal appearance of disease mayindicate the presence of a viral co-factor. In human cys-tic fibrosis (CF) patients, a seasonal pattern for acute* Correspondence: charlottemarksoerensen@hotmail.com1National Veterinary Institute, Technical University of Denmark, Bülowsvej 27,DK-1870, Frederiksberg C, DenmarkFull list of author information is available at the end of the article© 2012 Salomonsen et al.; licensee BioMed CeCreative Commons Attribution License (http:/distribution, and reproduction in any mediumand chronic P. aeruginosa infection has been identified[4] and the authors proposed respiratory viruses as theunderlying cause for this pattern. Respiratory viruseshave a seasonal infection pattern coinciding with that ofP. aeruginosa infection in CF patients and respiratoryviruses are known to precede bacterial infections [5,6].Respiratory syncytial virus (RSV) is an envelopedsingle-stranded negative sense RNA virus belonging tothe genus Pneumovirus in the family Paramyxoviridae.Variants of the virus infect various species includingsheep, goats, mice, cattle and humans. A number of ani-mal species can be experimentally infected with humanRSV (HRSV) [7,8]. The occurrence of HRSV infectionsin humans is known to be highly seasonal with the ma-jority of cases occurring in the winter or spring [9].HRSV has been shown to promote adhesion of P. aeru-ginosa to cells in the respiratory tract of mice [10,11]and to directly mediate binding of P. aeruginosa tohuman epithelial cells in vitro [12]. HRSV has also beenshown to replicate in mink lung cells in vitro [13] and inntral Ltd. This is an Open Access article distributed under the terms of the/creativecommons.org/licenses/by/2.0), which permits unrestricted use,, provided the original work is properly cited.Salomonsen et al. Acta Veterinaria Scandinavica 2012, 54:70 Page 2 of 3http://www.actavetscand.com/content/54/1/70the lungs of infant ferrets [14]. RSV has never beendescribed as naturally occurring in mink and experimen-tal studies on RSV infections in mink has not been pub-lished. However mink may be exposed to HRSV by theirhuman caretakers and to bovine RSV (BRSV) from thefeed which often include slaughter offal from cattle.The aim of the present study was to investigatewhether HRSV or BRSV could be found in lung tissuefrom mink succumbing to hemorrhagic pneumoniacaused by P. aeruginosa and hence possibly be part ofthe pathogenesis of this disease.Fifty samples of lung tissue from Danish mink wereselected of which 40 originated from mink submittedfrom 10 fur farms for routine diagnostic examinationand found to have hemorrhagic pneumonia associatedwith culturing of P. aeruginosa in pure culture from thelungs. Twenty of these mink originated from three farmswith high mortality due to hemorrhagic pneumonia(above 15%) while 15 mink were submitted from fivefarms with low mortality (below 2%). Data on mortalitywas not available for the last five mink submitted forroutine diagnostic investigation. Ten samples originatedfrom normal wildmink experimentally infected intra-nasally with 103-109 viable P. aeruginosa bacteria. Twoof the ten experimentally infected mink developed clin-ical symptoms of hemorrhagic pneumonia (lethargy,labored breathing) and were euthanized two days afterinoculation and necropsied immediately. The remainingeight clinically normal mink were euthanized eight daysafter inoculation and necropsied immediately. Theseeight mink served as negative controls. The animalexperiments were approved by the Danish AnimalExperiments Inspectorate. From the control mink, speci-mens of the left caudal lobe was obtained, while lung tis-sue with gross lesions of pneumonia was chosen fromthe experimentally infected clinically diseased mink andthe mink submitted for routine diagnostic. The sampleswere collected during 2011 and stored at −20°C untilfurther testing.Total RNA was purified from the lung tissue using theRNeasy Mini Kit (Qiagen, Düsseldorf, Germany) on aQIAcube (Qiagen) according to manufacturer’s instruc-tions. A BRSV positive cell culture was used for positivecontrol while nuclease-free water (Amresco, Solon, OH,USA) was used as a negative control of the purificationstep. The recovered RNA was stored at −80°C until fur-ther analysis.The presence of BRSV was tested with a modificationof a previously published conventional reverse tran-scriptase polymerase chain reaction (RT-PCR) assay tar-geting the F-gene with the forward primer sequence 50-AACCGGCTTCCTTCAGTAGAGC-30 and the reverseprimer sequence 50-CAATACCACCCACGATCTGTCC-30 [15]. The RT-PCR was performed on a Biometra T3Thermocycler (Biometra, Goettingen, Germany) in atotal volume of 25 μl using Qiagen OneStep RT-PCR kit(Qiagen) and 3 μl of extracted RNA, 400 μM dNTP mixand 500 nM of each primer. The amplificationtemperature profile was 30 min at 50°C for reverse tran-scription, 15 min at 95°C and 35 cycles of 30 s at 95°C,30 s at 55°C and 1 min at 72°C, followed by 10 min at72°C. In each PCR reaction a known BRSV positive sam-ple and a non-template control was run with the sam-ples as controls. PCR products with the expected size of730 bp were visualized on E-gelW 2% General PurposeAgarose (Invitrogen, Carlsbad, CA, USA).For detection of HRSV a commercially available real-time PCR (ProdesseW ProFlu™+, Gen-probe, San Diego,CA, USA) was employed. The primers used for this de-tection were not available due to the commercial natureof the PCR. The amplification and detection were per-formed on a MX3005p (Stratagene, La Jolla, CA, USA).The manufacturer’s protocol was followed in detail.The 50 mink lung samples were negative when testedin the BRSV and HRSV specific assays. In 5 samplestested with the BRSV specific primers, weak PCR pro-ducts of the predicted size were recognized but sequenceanalysis showed no resemblance to the F gene of BRSVor to other known genes for these products. Therefore,they probably represent unspecific annealing to minkspecific nucleic acids. No PCR products were obtainedusing the HRSV specific assay.Evidence of concurrent infection with HRSV andBRSV and P. aeruginosa in cases of mink hemorrhagicpneumonia was not found. Whether this was due tobreakdown of the virus, e.g. due to severe inflammation,collection of samples after clearance of the virus, or be-cause HRSV or BRSV simply was not present in the lungsamples remains uncertain.Histological examinations of lungs from mink suffer-ing from hemorrhagic pneumonia have not revealed syn-cytial cells [16,17] but due to the severe lung lesionsseen in this type of infection, syncytial cells may be diffi-cult to recognize or may be necrotic at the time of histo-logical examination. RSV can experimentally infect anumber of species [8] but is apparently quite host-specific in eliciting disease even though the various RSVsare closely related [7]. If a specific mink RSV is existingand is associated with hemorrhagic pneumonia in mink,the primers used for detection of BRSV and HRSV inthis study may not be able to detect this virus. To fur-ther elucidate whether a novel RSV-like virus is presentin mink, other primer-sets may be generated based onhighly conserved parts of genomes belonging to thegenus Pneumovirus. Electron microscopy, immunohisto-chemistry of lungs and nasal epithelium or de novo se-quencing of tissue from animals showing acuterespiratory symptoms may also be a valuable tool forSalomonsen et al. Acta Veterinaria Scandinavica 2012, 54:70 Page 3 of 3http://www.actavetscand.com/content/54/1/70identifying unrecognized respiratory viruses that may actas co-factors to P. aeruginosa hemorrhagic pneumonia.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsCMS participated in the design of the study, identified and collected suitablematerial, coordinated the study and drafted the manuscript. SØB participatedin the design of the study, supervised the BRSV testing, interpreted thesequencing of the weak PCR products and revised the manuscript. LELcontributed to the study design and revised the manuscript. JJ planned theHRSV testing and performed the practical experiment. NH contributed to thestudy design. ASH participated in the design of the study and the initialidentification of tissue used in the study. All authors read and approved thefinal manuscript.AcknowledgementsThe authors wish to thank Kirstine Vorborg for excellent technical assistance.Nordvacc and Kopenhagen Fur are thanked for financial support of thisstudy. The funding sources were not involved in study design, datacollection, analysis, interpretation, writing or decision to publish the workpresented in this communication.Author details1National Veterinary Institute, Technical University of Denmark, Bülowsvej 27,DK-1870, Frederiksberg C, Denmark. 2Department of Clinical Microbiology,Rigshospitalet, Blegdamsvej 9, DK-2100, Copenhagen Ø, Denmark.3Department of Veterinary Disease Biology, Faculty of Health and MedicalSciences, University of Copenhagen, Ridebanevej 3, DK-1870, Frederiksberg C,Denmark.Received: 30 July 2012 Accepted: 19 November 2012Published: 26 November 2012References1. Knox B: Pseudomonas aeruginosa as a cause of enzootic infections inmink [in Danish]. Nord Vet Med 1953, 5:731.2. Honda E, Homma JY, Abe C, Tanamoto K, Noda H, Yanagawa R: Effects ofthe common protective antigen (OEP) and toxoids of protease andelastase from Pseudomonas aeruginosa on protection againsthemorrhagic pneumonia in mink. Zentralbl Bakteriol [Orig A] 1977,237:297–309.3. Homma JY, Abe C, Tanamoto K, Hirao Y, Morihara K, Tsuzuki H, Yanagawa R,Honda E, Aoi Y, Fujimoto Y, Goryo M, Imazeki N, Noda H, Goda A, TakeuchiS, Ishihara T: Effectiveness of immunization with single and multi-component vaccines prepared from a common antigen (OEP), proteaseand elastase toxoids of Pseudomonas aeruginosa on protection againsthemorrhagic pneumonia in mink due to P. aeruginosa. Jpn J Exp Med1978, 48:111–133.4. Johansen HK, Hoiby N: Seasonal onset of initial colonisation and chronicinfection with Pseudomonas aeruginosa in patients with cystic fibrosis inDenmark. Thorax 1992, 47:109–111.5. Hament JM, Kimpen JL, Fleer A, Wolfs TF: Respiratory viral infectionpredisposing for bacterial disease: a concise review. FEMS Immunol MedMicrobiol 1999, 26:189–195.6. Avadhanula V, Rodriguez CA, Devincenzo JP, Wang Y, Webby RJ, Ulett GC,Adderson EE: Respiratory viruses augment the adhesion of bacterialpathogens to respiratory epithelium in a viral species- and cell type-dependent manner. J Virol 2006, 80:1629–1636.7. Easton AJ, Domachowske JB, Rosenberg HF: Animal pneumoviruses:molecular genetics and pathogenesis. Clin Microbiol Rev 2004, 17:390–412.8. Byrd LG, Prince GA: Animal models of respiratory syncytial virus infection.Clin Infect Dis 1997, 25:1363–1368.9. Centers for Disease Control and Prevention (CDC): Brief report: respiratorysyncytial virus activity - United States, 2005–2006. MMWR Morb MortalWkly Rep 2006, 55:1277–1279.10. Stark JM, Stark MA, Colasurdo GN, LeVine AM: Decreased bacterialclearance from the lungs of mice following primary respiratory syncytialvirus infection. J Med Virol 2006, 78:829–838.11. de Vrankrijker AM, Wolfs TF, Ciofu O, Hoiby N, van der Ent CK, Poulsen SS,Johansen HK: Respiratory syncytial virus infection facilitates acutecolonization of Pseudomonas aeruginosa in mice. J Med Virol 2009,81:2096–2103.12. Van Ewijk BE, Wolfs TF, Aerts PC, Van Kessel KP, Fleer A, Kimpen JL, Van derEnt CK: RSV mediates Pseudomonas aeruginosa binding to cystic fibrosisand normal epithelial cells. Pediatr Res 2007, 61:398–403.13. Yeolekar LR, Damle RG, Basu A, Rao BL: Growth of respiratory syncytialvirus in mink lung epithelial cells. Indian J Med Res 2002, 116:255–257.14. Prince GA, Porter DD: The pathogenesis of respiratory syncytial virusinfection in infant ferrets. Am J Pathol 1976, 82:339–352.15. Larsen LE, Tjornehoj K, Viuff B, Jensen NE, Uttenthal A: Diagnosis ofenzootic pneumonia in Danish cattle: reverse transcription-polymerasechain reaction assay for detection of bovine respiratory syncytial virus innaturally and experimentally infected cattle. J Vet Diagn Invest 1999,11:416–422.16. Nordstoga K: Pseudomonas infections in mink with special reference topseudomonas vasculitis in pulmonary lesions. Acta Vet Scand 1968, 9:33–40.17. Long GG, Gallina AM, Gorham JR: Pseudomonas pneumonia of mink:pathogenesis, vaccination, and serologic studies. Am J Vet Res 1980,41:1720–1725.doi:10.1186/1751-0147-54-70Cite this article as: Salomonsen et al.: Investigation of the presence ofhuman or bovine respiratory syncytial virus in the lungs of mink(Neovison vison) with hemorrhagic pneumonia due to Pseudomonasaeruginosa. Acta Veterinaria Scandinavica 2012 54:70.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color ﬁgure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submit

BackgroundHemorrhagic pneumonia is a disease of farmed mink (Neovison vison) caused by Pseudomonas aeruginosa. The disease is highly seasonal in Danish mink with outbreaks occurring almost exclusively in the autumn. Human respiratory syncytial virus (RSV) has been shown to augment infection with P. aeruginosa in mice and to promote adhesion of P. aeruginosa to human respiratory cells.FindingsWe tested 50 lung specimens from mink with hemorrhagic pneumonia for bovine RSV by reverse transcriptase polymerase chain reaction (PCR) and for human RSV by a commercial real-time PCR. RSV was not found.ConclusionsThis study indicates that human and bovine RSV is not a major co-factor for development of hemorrhagic pneumonia in Danish mink

Hammer , Anne Sofie

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Downloaded from orbit.dtu.dk on: Dec 20, 2017Investigation of the presence of human or bovine respiratory syncytial virus in thelungs of mink (Neovison vison) with hemorrhagic pneumonia due to PseudomonasaeruginosaSalomonsen, Charlotte Mark; Breum, Solvej Østergaard; Larsen, Lars Erik; Jakobsen, Jeanette; Høiby,Niels; Hammer , Anne SofiePublished in:Acta Veterinaria Scandinavica (Online)Link to article, DOI:10.1186/1751-0147-54-70Publication date:2012Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Salomonsen, C. M., Breum, S. Ø., Larsen, L. E., Jakobsen, J., Høiby, N., & Hammer , A. S. (2012). Investigationof the presence of human or bovine respiratory syncytial virus in the lungs of mink (Neovison vison) withhemorrhagic pneumonia due to Pseudomonas aeruginosa. Acta Veterinaria Scandinavica (Online), 54(70). DOI:10.1186/1751-0147-54-70BRIEF COMMUNICATION Open AccessInvestigation of the presence of human or bovinerespiratory syncytial virus in the lungs of mink(Neovison vison) with hemorrhagic pneumoniadue to Pseudomonas aeruginosaCharlotte M Salomonsen1*, Solvej Ø Breum1, Lars E Larsen1, Jeanette Jakobsen2, Niels Høiby2and Anne S Hammer1,3AbstractBackground: Hemorrhagic pneumonia is a disease of farmed mink (Neovison vison) caused by Pseudomonasaeruginosa. The disease is highly seasonal in Danish mink with outbreaks occurring almost exclusively in theautumn. Human respiratory syncytial virus (RSV) has been shown to augment infection with P. aeruginosa in miceand to promote adhesion of P. aeruginosa to human respiratory cells.Findings: We tested 50 lung specimens from mink with hemorrhagic pneumonia for bovine RSV by reversetranscriptase polymerase chain reaction (PCR) and for human RSV by a commercial real-time PCR. RSV was notfound.Conclusions: This study indicates that human and bovine RSV is not a major co-factor for development ofhemorrhagic pneumonia in Danish mink.Keywords: Hemorrhagic pneumonia, Mink, Pseudomonas aeruginosa, Respiratory syncytial virusFindingsPseudomonas aeruginosa was identified as the cause ofhemorrhagic pneumonia in farmed mink (Neovisonvison) in 1953 [1]. This disease can cause mortalities ashigh as 50% on the farm level [2,3] but can also follow amilder course [1]. Hemorrhagic pneumonia is character-ized by a rapid disease progression in the individualmink and it is highly seasonal in Denmark with farmoutbreaks occurring almost exclusively from Septemberto November. Whether the seasonal pattern is related tothe seasonal life cycle of farmed mink or if it relates toco-infection with other pathogens has not been investi-gated. Thus, no infectious co-factors have been identi-fied but the highly seasonal appearance of disease mayindicate the presence of a viral co-factor. In human cys-tic fibrosis (CF) patients, a seasonal pattern for acuteand chronic P. aeruginosa infection has been identified[4] and the authors proposed respiratory viruses as theunderlying cause for this pattern. Respiratory viruseshave a seasonal infection pattern coinciding with that ofP. aeruginosa infection in CF patients and respiratoryviruses are known to precede bacterial infections [5,6].Respiratory syncytial virus (RSV) is an envelopedsingle-stranded negative sense RNA virus belonging tothe genus Pneumovirus in the family Paramyxoviridae.Variants of the virus infect various species includingsheep, goats, mice, cattle and humans. A number of ani-mal species can be experimentally infected with humanRSV (HRSV) [7,8]. The occurrence of HRSV infectionsin humans is known to be highly seasonal with the ma-jority of cases occurring in the winter or spring [9].HRSV has been shown to promote adhesion of P. aeru-ginosa to cells in the respiratory tract of mice [10,11]and to directly mediate binding of P. aeruginosa tohuman epithelial cells in vitro [12]. HRSV has also beenshown to replicate in mink lung cells in vitro [13] and in* Correspondence: charlottemarksoerensen@hotmail.com1National Veterinary Institute, Technical University of Denmark, Bülowsvej 27,DK-1870, Frederiksberg C, DenmarkFull list of author information is available at the end of the article© 2012 Salomonsen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.Salomonsen et al. Acta Veterinaria Scandinavica 2012, 54:70http://www.actavetscand.com/content/54/1/70the lungs of infant ferrets [14]. RSV has never beendescribed as naturally occurring in mink and experimen-tal studies on RSV infections in mink has not been pub-lished. However mink may be exposed to HRSV by theirhuman caretakers and to bovine RSV (BRSV) from thefeed which often include slaughter offal from cattle.The aim of the present study was to investigatewhether HRSV or BRSV could be found in lung tissuefrom mink succumbing to hemorrhagic pneumoniacaused by P. aeruginosa and hence possibly be part ofthe pathogenesis of this disease.Fifty samples of lung tissue from Danish mink wereselected of which 40 originated from mink submittedfrom 10 fur farms for routine diagnostic examinationand found to have hemorrhagic pneumonia associatedwith culturing of P. aeruginosa in pure culture from thelungs. Twenty of these mink originated from three farmswith high mortality due to hemorrhagic pneumonia(above 15%) while 15 mink were submitted from fivefarms with low mortality (below 2%). Data on mortalitywas not available for the last five mink submitted forroutine diagnostic investigation. Ten samples originatedfrom normal wildmink experimentally infected intra-nasally with 103-109 viable P. aeruginosa bacteria. Twoof the ten experimentally infected mink developed clin-ical symptoms of hemorrhagic pneumonia (lethargy,labored breathing) and were euthanized two days afterinoculation and necropsied immediately. The remainingeight clinically normal mink were euthanized eight daysafter inoculation and necropsied immediately. Theseeight mink served as negative controls. The animalexperiments were approved by the Danish AnimalExperiments Inspectorate. From the control mink, speci-mens of the left caudal lobe was obtained, while lung tis-sue with gross lesions of pneumonia was chosen fromthe experimentally infected clinically diseased mink andthe mink submitted for routine diagnostic. The sampleswere collected during 2011 and stored at −20°C untilfurther testing.Total RNA was purified from the lung tissue using theRNeasy Mini Kit (Qiagen, Düsseldorf, Germany) on aQIAcube (Qiagen) according to manufacturer’s instruc-tions. A BRSV positive cell culture was used for positivecontrol while nuclease-free water (Amresco, Solon, OH,USA) was used as a negative control of the purificationstep. The recovered RNA was stored at −80°C until fur-ther analysis.The presence of BRSV was tested with a modificationof a previously published conventional reverse tran-scriptase polymerase chain reaction (RT-PCR) assay tar-geting the F-gene with the forward primer sequence 50-AACCGGCTTCCTTCAGTAGAGC-30 and the reverseprimer sequence 50-CAATACCACCCACGATCTGTCC-30 [15]. The RT-PCR was performed on a Biometra T3Thermocycler (Biometra, Goettingen, Germany) in atotal volume of 25 μl using Qiagen OneStep RT-PCR kit(Qiagen) and 3 μl of extracted RNA, 400 μM dNTP mixand 500 nM of each primer. The amplificationtemperature profile was 30 min at 50°C for reverse tran-scription, 15 min at 95°C and 35 cycles of 30 s at 95°C,30 s at 55°C and 1 min at 72°C, followed by 10 min at72°C. In each PCR reaction a known BRSV positive sam-ple and a non-template control was run with the sam-ples as controls. PCR products with the expected size of730 bp were visualized on E-gelW 2% General PurposeAgarose (Invitrogen, Carlsbad, CA, USA).For detection of HRSV a commercially available real-time PCR (ProdesseW ProFlu™+, Gen-probe, San Diego,CA, USA) was employed. The primers used for this de-tection were not available due to the commercial natureof the PCR. The amplification and detection were per-formed on a MX3005p (Stratagene, La Jolla, CA, USA).The manufacturer’s protocol was followed in detail.The 50 mink lung samples were negative when testedin the BRSV and HRSV specific assays. In 5 samplestested with the BRSV specific primers, weak PCR pro-ducts of the predicted size were recognized but sequenceanalysis showed no resemblance to the F gene of BRSVor to other known genes for these products. Therefore,they probably represent unspecific annealing to minkspecific nucleic acids. No PCR products were obtainedusing the HRSV specific assay.Evidence of concurrent infection with HRSV andBRSV and P. aeruginosa in cases of mink hemorrhagicpneumonia was not found. Whether this was due tobreakdown of the virus, e.g. due to severe inflammation,collection of samples after clearance of the virus, or be-cause HRSV or BRSV simply was not present in the lungsamples remains uncertain.Histological examinations of lungs from mink suffer-ing from hemorrhagic pneumonia have not revealed syn-cytial cells [16,17] but due to the severe lung lesionsseen in this type of infection, syncytial cells may be diffi-cult to recognize or may be necrotic at the time of histo-logical examination. RSV can experimentally infect anumber of species [8] but is apparently quite host-specific in eliciting disease even though the various RSVsare closely related [7]. If a specific mink RSV is existingand is associated with hemorrhagic pneumonia in mink,the primers used for detection of BRSV and HRSV inthis study may not be able to detect this virus. To fur-ther elucidate whether a novel RSV-like virus is presentin mink, other primer-sets may be generated based onhighly conserved parts of genomes belonging to thegenus Pneumovirus. Electron microscopy, immunohisto-chemistry of lungs and nasal epithelium or de novo se-quencing of tissue from animals showing acuterespiratory symptoms may also be a valuable tool forSalomonsen et al. Acta Veterinaria Scandinavica 2012, 54:70 Page 2 of 3http://www.actavetscand.com/content/54/1/70identifying unrecognized respiratory viruses that may actas co-factors to P. aeruginosa hemorrhagic pneumonia.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsCMS participated in the design of the study, identified and collected suitablematerial, coordinated the study and drafted the manuscript. SØB participatedin the design of the study, supervised the BRSV testing, interpreted thesequencing of the weak PCR products and revised the manuscript. LELcontributed to the study design and revised the manuscript. JJ planned theHRSV testing and performed the practical experiment. NH contributed to thestudy design. ASH participated in the design of the study and the initialidentification of tissue used in the study. All authors read and approved thefinal manuscript.AcknowledgementsThe authors wish to thank Kirstine Vorborg for excellent technical assistance.Nordvacc and Kopenhagen Fur are thanked for financial support of thisstudy. The funding sources were not involved in study design, datacollection, analysis, interpretation, writing or decision to publish the workpresented in this communication.Author details1National Veterinary Institute, Technical University of Denmark, Bülowsvej 27,DK-1870, Frederiksberg C, Denmark. 2Department of Clinical Microbiology,Rigshospitalet, Blegdamsvej 9, DK-2100, Copenhagen Ø, Denmark.3Department of Veterinary Disease Biology, Faculty of Health and MedicalSciences, University of Copenhagen, Ridebanevej 3, DK-1870, Frederiksberg C,Denmark.Received: 30 July 2012 Accepted: 19 November 2012Published: 26 November 2012References1. Knox B: Pseudomonas aeruginosa as a cause of enzootic infections inmink [in Danish]. Nord Vet Med 1953, 5:731.2. Honda E, Homma JY, Abe C, Tanamoto K, Noda H, Yanagawa R: Effects ofthe common protective antigen (OEP) and toxoids of protease andelastase from Pseudomonas aeruginosa on protection againsthemorrhagic pneumonia in mink. Zentralbl Bakteriol [Orig A] 1977,237:297–309.3. Homma JY, Abe C, Tanamoto K, Hirao Y, Morihara K, Tsuzuki H, Yanagawa R,Honda E, Aoi Y, Fujimoto Y, Goryo M, Imazeki N, Noda H, Goda A, TakeuchiS, Ishihara T: Effectiveness of immunization with single and multi-component vaccines prepared from a common antigen (OEP), proteaseand elastase toxoids of Pseudomonas aeruginosa on protection againsthemorrhagic pneumonia in mink due to P. aeruginosa. Jpn J Exp Med1978, 48:111–133.4. Johansen HK, Hoiby N: Seasonal onset of initial colonisation and chronicinfection with Pseudomonas aeruginosa in patients with cystic fibrosis inDenmark. Thorax 1992, 47:109–111.5. 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Larsen LE, Tjornehoj K, Viuff B, Jensen NE, Uttenthal A: Diagnosis ofenzootic pneumonia in Danish cattle: reverse transcription-polymerasechain reaction assay for detection of bovine respiratory syncytial virus innaturally and experimentally infected cattle. J Vet Diagn Invest 1999,11:416–422.16. Nordstoga K: Pseudomonas infections in mink with special reference topseudomonas vasculitis in pulmonary lesions. Acta Vet Scand 1968, 9:33–40.17. Long GG, Gallina AM, Gorham JR: Pseudomonas pneumonia of mink:pathogenesis, vaccination, and serologic studies. Am J Vet Res 1980,41:1720–1725.doi:10.1186/1751-0147-54-70Cite this article as: Salomonsen et al.: Investigation of the presence ofhuman or bovine respiratory syncytial virus in the lungs of mink(Neovison vison) with hemorrhagic pneumonia due to Pseudomonasaeruginosa. Acta Veterinaria Scandinavica 2012 54:70.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color ﬁgure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitSalomonsen et al. Acta Veterinaria Scandinavica 2012, 54:70 Page 3 of 3http://www.actavetscand.com/content/54/1/70

Adderson EE: Respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species- and cell typedependent manner.

AM: Decreased bacterial clearance from the lungs of mice following primary respiratory syncytial virus infection.

DD: The pathogenesis of respiratory syncytial virus infection in infant ferrets.

Disease Control and Prevention (CDC): Brief report: respiratory syncytial virus activity - United States, 2005–2006. MMWR Morb Mortal Wkly Rep

Effects of the common protective antigen (OEP) and toxoids of protease and elastase from Pseudomonas aeruginosa on protection against hemorrhagic pneumonia in mink. Zentralbl Bakteriol [Orig A]

HF: Animal pneumoviruses: molecular genetics and pathogenesis. Clin Microbiol Rev

HK: Respiratory syncytial virus infection facilitates acute colonization of Pseudomonas aeruginosa in mice.

Hoiby N: Seasonal onset of initial colonisation and chronic infection with Pseudomonas aeruginosa in patients with cystic fibrosis in Denmark. Thorax

Ishihara T: Effectiveness of immunization with single and multicomponent vaccines prepared from a common antigen (OEP), protease and elastase toxoids of Pseudomonas aeruginosa on protection against hemorrhagic pneumonia in mink due to P. aeruginosa.

Prince GA: Animal models of respiratory syncytial virus infection. Clin Infect Dis

Pseudomonas aeruginosa as a cause of enzootic infections in mink [in Danish]. Nord Vet Med

Pseudomonas infections in mink with special reference to pseudomonas vasculitis in pulmonary lesions. Acta Vet Scand

Rao BL: Growth of respiratory syncytial virus in mink lung epithelial cells.

Uttenthal A: Diagnosis of enzootic pneumonia in Danish cattle: reverse transcription-polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle. J Vet Diagn Invest

Van der Ent CK: RSV mediates Pseudomonas aeruginosa binding to cystic fibrosis and normal epithelial cells. Pediatr Res

Wolfs TF: Respiratory viral infection predisposing for bacterial disease: a concise review. FEMS Immunol Med Microbiol

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Investigation of the presence of human or bovine respiratory syncytial virus in the lungs of mink (Neovison vison) with hemorrhagic pneumonia due to Pseudomonas aeruginosa

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