Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge
Avian influenza (AI) is a significant public health concern and serious economic threat to the commercial poultry industry worldwide. Previous research demonstrates that antibodies against M2e confer protection against influenza challenge. Using the Red recombinase system in combination with overlap...
Gespeichert in:
Veröffentlicht in: | Poultry science 2009-11, Vol.88 (11), p.2244-2252 |
---|---|
Hauptverfasser: | , , , , , , , , , , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
container_end_page | 2252 |
---|---|
container_issue | 11 |
container_start_page | 2244 |
container_title | Poultry science |
container_volume | 88 |
creator | Layton, S.L Kapczynski, D.R Higgins, S Higgins, J Wolfenden, A.D Liljebjelke, K.A Bottje, W.G Swayne, D Berghman, L.R Kwon, Y.M Hargis, B.M Cole, K |
description | Avian influenza (AI) is a significant public health concern and serious economic threat to the commercial poultry industry worldwide. Previous research demonstrates that antibodies against M2e confer protection against influenza challenge. Using the Red recombinase system in combination with overlapping extension PCR, we recently developed several novel attenuated Salmonella Enteritidis strains that express a protective M2e epitope in combination with a potential immune-enhancing CD154 peptide sequence on the Salmonella outer membrane protein lamB. Commercial Leghorn chicks were orally immunized (immunization dose: 10⁶ to 10⁸ cfu/chick) with saline (negative control) or one of the recombinant Salmonella strains [ΔaroA M2e-CD154, ΔhtrA M2e-CD154, ΔaroA-ΔhtrA M2e(4)-CD154] on day of hatch and 21 d posthatch. These candidate vaccine strains were evaluated for their ability to invade, colonize, and persist in tissues and elicit an M2e-specific antibody response. The vaccine candidate strain ΔaroA M2e-CD154 exhibited significantly greater organ invasion in the liver and spleen at d 7 (P > 0.05); however, no marked differences in colonization of the cecal tonsils were observed. Vaccinated chickens exhibited significantly increased M2e-specific IgG responses, which were further enhanced by simultaneous expression of CD154 (P < 0.05). Virus neutralization assays gave neutralizing indices of 6.6, 6.3, and 6.3 for ΔaroA M2e-CD154, ΔhtrA M2e-CD154, and ΔaroA-ΔhtrA M2e(4)-CD154 seven days post booster immunization, respectively, indicating effective neutralization of AI by serum IgG of vaccinated chickens. In a subsequent direct challenge study, specific-pathogen-free Leghorn chicks immunized with ΔaroA-ΔhtrA M2e(4)-CD154 offered significant protection against direct challenge with low pathogenic AI H7N2, but not highly pathogenic H5N1 AI. Taken together, these data suggest that these Salmonella-vectored vaccines expressing M2e in association with CD154 are effective at protecting chickens against low pathogenic AI. |
doi_str_mv | 10.3382/ps.2009-00251 |
format | Article |
fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_734092145</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>734092145</sourcerecordid><originalsourceid>FETCH-LOGICAL-c355t-79583530bd9c5c57c7dc777f2bd8d9e6a859f5e93fc49345d236ebde1960ac3c3</originalsourceid><addsrcrecordid>eNpFkU1v1DAQhi0EokvhyBV845TijziOj2jLl1TEoZSr5diTXUNiB9vbAj-M34fTXcRpRppHj2bmReg5JRec9-z1ki8YIaohhAn6AG2oYKLhVNKHaEMIZ42Qip6hJzl_qwjtOvkYnVHV85ZItkF_vhprfTDFx4DjiO3e2-8QMr7zZY8T2DgPdRwKvjbTHANMk8Hwc0mQsw87_IkBNsHh7SUVLYbFl7hAxj7YBCbXbkmxgL3Xr5yDf4Nbn8yE8x6cW0VmLJDwFO_wYso-7iB4i82tN6HKxukA4bep25lpgrCDp-jRaKYMz071HN28e_tl-6G5-vz-4_bNVWO5EKWRSvRccDI4ZYUV0kpnpZQjG1zvFHSmF2oUoPhoW8Vb4RjvYHBAVUeM5Zafo1dHbz3jxwFy0bPPdn1CgHjIWtY3KkZbUcnmSNoUc04w6iX52aRfmhK9JqWXrNek9H1SlX9xMh-GGdx_-hRNBV4egdFEbXbJZ31zzQjlhHaqUoz_BXPrnE4</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>734092145</pqid></control><display><type>article</type><title>Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge</title><source>MEDLINE</source><source>Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals</source><source>Alma/SFX Local Collection</source><creator>Layton, S.L ; Kapczynski, D.R ; Higgins, S ; Higgins, J ; Wolfenden, A.D ; Liljebjelke, K.A ; Bottje, W.G ; Swayne, D ; Berghman, L.R ; Kwon, Y.M ; Hargis, B.M ; Cole, K</creator><creatorcontrib>Layton, S.L ; Kapczynski, D.R ; Higgins, S ; Higgins, J ; Wolfenden, A.D ; Liljebjelke, K.A ; Bottje, W.G ; Swayne, D ; Berghman, L.R ; Kwon, Y.M ; Hargis, B.M ; Cole, K</creatorcontrib><description>Avian influenza (AI) is a significant public health concern and serious economic threat to the commercial poultry industry worldwide. Previous research demonstrates that antibodies against M2e confer protection against influenza challenge. Using the Red recombinase system in combination with overlapping extension PCR, we recently developed several novel attenuated Salmonella Enteritidis strains that express a protective M2e epitope in combination with a potential immune-enhancing CD154 peptide sequence on the Salmonella outer membrane protein lamB. Commercial Leghorn chicks were orally immunized (immunization dose: 10⁶ to 10⁸ cfu/chick) with saline (negative control) or one of the recombinant Salmonella strains [ΔaroA M2e-CD154, ΔhtrA M2e-CD154, ΔaroA-ΔhtrA M2e(4)-CD154] on day of hatch and 21 d posthatch. These candidate vaccine strains were evaluated for their ability to invade, colonize, and persist in tissues and elicit an M2e-specific antibody response. The vaccine candidate strain ΔaroA M2e-CD154 exhibited significantly greater organ invasion in the liver and spleen at d 7 (P > 0.05); however, no marked differences in colonization of the cecal tonsils were observed. Vaccinated chickens exhibited significantly increased M2e-specific IgG responses, which were further enhanced by simultaneous expression of CD154 (P < 0.05). Virus neutralization assays gave neutralizing indices of 6.6, 6.3, and 6.3 for ΔaroA M2e-CD154, ΔhtrA M2e-CD154, and ΔaroA-ΔhtrA M2e(4)-CD154 seven days post booster immunization, respectively, indicating effective neutralization of AI by serum IgG of vaccinated chickens. In a subsequent direct challenge study, specific-pathogen-free Leghorn chicks immunized with ΔaroA-ΔhtrA M2e(4)-CD154 offered significant protection against direct challenge with low pathogenic AI H7N2, but not highly pathogenic H5N1 AI. Taken together, these data suggest that these Salmonella-vectored vaccines expressing M2e in association with CD154 are effective at protecting chickens against low pathogenic AI.</description><identifier>ISSN: 0032-5791</identifier><identifier>EISSN: 1525-3171</identifier><identifier>DOI: 10.3382/ps.2009-00251</identifier><identifier>PMID: 19834072</identifier><language>eng</language><publisher>England: Poultry Science Association</publisher><subject>Animals ; Antigens, Viral - genetics ; Antigens, Viral - immunology ; Antigens, Viral - metabolism ; avian influenza ; Chickens ; Epitopes - genetics ; Epitopes - metabolism ; immune response ; immunoglobulin G ; Influenza A virus ; Influenza in Birds - prevention & control ; Influenza Vaccines - immunology ; liver ; membrane proteins ; microbial colonization ; molecular sequence data ; neutralization tests ; nucleotide sequences ; oral administration ; pathogenicity ; polymerase chain reaction ; recombinant antigens ; recombinant vaccines ; Salmonella - genetics ; Salmonella - metabolism ; Salmonella enteritidis ; spleen ; strains ; tonsils ; vaccination ; viral antigens ; Virus Shedding</subject><ispartof>Poultry science, 2009-11, Vol.88 (11), p.2244-2252</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c355t-79583530bd9c5c57c7dc777f2bd8d9e6a859f5e93fc49345d236ebde1960ac3c3</citedby><cites>FETCH-LOGICAL-c355t-79583530bd9c5c57c7dc777f2bd8d9e6a859f5e93fc49345d236ebde1960ac3c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19834072$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Layton, S.L</creatorcontrib><creatorcontrib>Kapczynski, D.R</creatorcontrib><creatorcontrib>Higgins, S</creatorcontrib><creatorcontrib>Higgins, J</creatorcontrib><creatorcontrib>Wolfenden, A.D</creatorcontrib><creatorcontrib>Liljebjelke, K.A</creatorcontrib><creatorcontrib>Bottje, W.G</creatorcontrib><creatorcontrib>Swayne, D</creatorcontrib><creatorcontrib>Berghman, L.R</creatorcontrib><creatorcontrib>Kwon, Y.M</creatorcontrib><creatorcontrib>Hargis, B.M</creatorcontrib><creatorcontrib>Cole, K</creatorcontrib><title>Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge</title><title>Poultry science</title><addtitle>Poult Sci</addtitle><description>Avian influenza (AI) is a significant public health concern and serious economic threat to the commercial poultry industry worldwide. Previous research demonstrates that antibodies against M2e confer protection against influenza challenge. Using the Red recombinase system in combination with overlapping extension PCR, we recently developed several novel attenuated Salmonella Enteritidis strains that express a protective M2e epitope in combination with a potential immune-enhancing CD154 peptide sequence on the Salmonella outer membrane protein lamB. Commercial Leghorn chicks were orally immunized (immunization dose: 10⁶ to 10⁸ cfu/chick) with saline (negative control) or one of the recombinant Salmonella strains [ΔaroA M2e-CD154, ΔhtrA M2e-CD154, ΔaroA-ΔhtrA M2e(4)-CD154] on day of hatch and 21 d posthatch. These candidate vaccine strains were evaluated for their ability to invade, colonize, and persist in tissues and elicit an M2e-specific antibody response. The vaccine candidate strain ΔaroA M2e-CD154 exhibited significantly greater organ invasion in the liver and spleen at d 7 (P > 0.05); however, no marked differences in colonization of the cecal tonsils were observed. Vaccinated chickens exhibited significantly increased M2e-specific IgG responses, which were further enhanced by simultaneous expression of CD154 (P < 0.05). Virus neutralization assays gave neutralizing indices of 6.6, 6.3, and 6.3 for ΔaroA M2e-CD154, ΔhtrA M2e-CD154, and ΔaroA-ΔhtrA M2e(4)-CD154 seven days post booster immunization, respectively, indicating effective neutralization of AI by serum IgG of vaccinated chickens. In a subsequent direct challenge study, specific-pathogen-free Leghorn chicks immunized with ΔaroA-ΔhtrA M2e(4)-CD154 offered significant protection against direct challenge with low pathogenic AI H7N2, but not highly pathogenic H5N1 AI. Taken together, these data suggest that these Salmonella-vectored vaccines expressing M2e in association with CD154 are effective at protecting chickens against low pathogenic AI.</description><subject>Animals</subject><subject>Antigens, Viral - genetics</subject><subject>Antigens, Viral - immunology</subject><subject>Antigens, Viral - metabolism</subject><subject>avian influenza</subject><subject>Chickens</subject><subject>Epitopes - genetics</subject><subject>Epitopes - metabolism</subject><subject>immune response</subject><subject>immunoglobulin G</subject><subject>Influenza A virus</subject><subject>Influenza in Birds - prevention & control</subject><subject>Influenza Vaccines - immunology</subject><subject>liver</subject><subject>membrane proteins</subject><subject>microbial colonization</subject><subject>molecular sequence data</subject><subject>neutralization tests</subject><subject>nucleotide sequences</subject><subject>oral administration</subject><subject>pathogenicity</subject><subject>polymerase chain reaction</subject><subject>recombinant antigens</subject><subject>recombinant vaccines</subject><subject>Salmonella - genetics</subject><subject>Salmonella - metabolism</subject><subject>Salmonella enteritidis</subject><subject>spleen</subject><subject>strains</subject><subject>tonsils</subject><subject>vaccination</subject><subject>viral antigens</subject><subject>Virus Shedding</subject><issn>0032-5791</issn><issn>1525-3171</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpFkU1v1DAQhi0EokvhyBV845TijziOj2jLl1TEoZSr5diTXUNiB9vbAj-M34fTXcRpRppHj2bmReg5JRec9-z1ki8YIaohhAn6AG2oYKLhVNKHaEMIZ42Qip6hJzl_qwjtOvkYnVHV85ZItkF_vhprfTDFx4DjiO3e2-8QMr7zZY8T2DgPdRwKvjbTHANMk8Hwc0mQsw87_IkBNsHh7SUVLYbFl7hAxj7YBCbXbkmxgL3Xr5yDf4Nbn8yE8x6cW0VmLJDwFO_wYso-7iB4i82tN6HKxukA4bep25lpgrCDp-jRaKYMz071HN28e_tl-6G5-vz-4_bNVWO5EKWRSvRccDI4ZYUV0kpnpZQjG1zvFHSmF2oUoPhoW8Vb4RjvYHBAVUeM5Zafo1dHbz3jxwFy0bPPdn1CgHjIWtY3KkZbUcnmSNoUc04w6iX52aRfmhK9JqWXrNek9H1SlX9xMh-GGdx_-hRNBV4egdFEbXbJZ31zzQjlhHaqUoz_BXPrnE4</recordid><startdate>20091101</startdate><enddate>20091101</enddate><creator>Layton, S.L</creator><creator>Kapczynski, D.R</creator><creator>Higgins, S</creator><creator>Higgins, J</creator><creator>Wolfenden, A.D</creator><creator>Liljebjelke, K.A</creator><creator>Bottje, W.G</creator><creator>Swayne, D</creator><creator>Berghman, L.R</creator><creator>Kwon, Y.M</creator><creator>Hargis, B.M</creator><creator>Cole, K</creator><general>Poultry Science Association</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20091101</creationdate><title>Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge</title><author>Layton, S.L ; Kapczynski, D.R ; Higgins, S ; Higgins, J ; Wolfenden, A.D ; Liljebjelke, K.A ; Bottje, W.G ; Swayne, D ; Berghman, L.R ; Kwon, Y.M ; Hargis, B.M ; Cole, K</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c355t-79583530bd9c5c57c7dc777f2bd8d9e6a859f5e93fc49345d236ebde1960ac3c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>Animals</topic><topic>Antigens, Viral - genetics</topic><topic>Antigens, Viral - immunology</topic><topic>Antigens, Viral - metabolism</topic><topic>avian influenza</topic><topic>Chickens</topic><topic>Epitopes - genetics</topic><topic>Epitopes - metabolism</topic><topic>immune response</topic><topic>immunoglobulin G</topic><topic>Influenza A virus</topic><topic>Influenza in Birds - prevention & control</topic><topic>Influenza Vaccines - immunology</topic><topic>liver</topic><topic>membrane proteins</topic><topic>microbial colonization</topic><topic>molecular sequence data</topic><topic>neutralization tests</topic><topic>nucleotide sequences</topic><topic>oral administration</topic><topic>pathogenicity</topic><topic>polymerase chain reaction</topic><topic>recombinant antigens</topic><topic>recombinant vaccines</topic><topic>Salmonella - genetics</topic><topic>Salmonella - metabolism</topic><topic>Salmonella enteritidis</topic><topic>spleen</topic><topic>strains</topic><topic>tonsils</topic><topic>vaccination</topic><topic>viral antigens</topic><topic>Virus Shedding</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Layton, S.L</creatorcontrib><creatorcontrib>Kapczynski, D.R</creatorcontrib><creatorcontrib>Higgins, S</creatorcontrib><creatorcontrib>Higgins, J</creatorcontrib><creatorcontrib>Wolfenden, A.D</creatorcontrib><creatorcontrib>Liljebjelke, K.A</creatorcontrib><creatorcontrib>Bottje, W.G</creatorcontrib><creatorcontrib>Swayne, D</creatorcontrib><creatorcontrib>Berghman, L.R</creatorcontrib><creatorcontrib>Kwon, Y.M</creatorcontrib><creatorcontrib>Hargis, B.M</creatorcontrib><creatorcontrib>Cole, K</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Poultry science</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Layton, S.L</au><au>Kapczynski, D.R</au><au>Higgins, S</au><au>Higgins, J</au><au>Wolfenden, A.D</au><au>Liljebjelke, K.A</au><au>Bottje, W.G</au><au>Swayne, D</au><au>Berghman, L.R</au><au>Kwon, Y.M</au><au>Hargis, B.M</au><au>Cole, K</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge</atitle><jtitle>Poultry science</jtitle><addtitle>Poult Sci</addtitle><date>2009-11-01</date><risdate>2009</risdate><volume>88</volume><issue>11</issue><spage>2244</spage><epage>2252</epage><pages>2244-2252</pages><issn>0032-5791</issn><eissn>1525-3171</eissn><abstract>Avian influenza (AI) is a significant public health concern and serious economic threat to the commercial poultry industry worldwide. Previous research demonstrates that antibodies against M2e confer protection against influenza challenge. Using the Red recombinase system in combination with overlapping extension PCR, we recently developed several novel attenuated Salmonella Enteritidis strains that express a protective M2e epitope in combination with a potential immune-enhancing CD154 peptide sequence on the Salmonella outer membrane protein lamB. Commercial Leghorn chicks were orally immunized (immunization dose: 10⁶ to 10⁸ cfu/chick) with saline (negative control) or one of the recombinant Salmonella strains [ΔaroA M2e-CD154, ΔhtrA M2e-CD154, ΔaroA-ΔhtrA M2e(4)-CD154] on day of hatch and 21 d posthatch. These candidate vaccine strains were evaluated for their ability to invade, colonize, and persist in tissues and elicit an M2e-specific antibody response. The vaccine candidate strain ΔaroA M2e-CD154 exhibited significantly greater organ invasion in the liver and spleen at d 7 (P > 0.05); however, no marked differences in colonization of the cecal tonsils were observed. Vaccinated chickens exhibited significantly increased M2e-specific IgG responses, which were further enhanced by simultaneous expression of CD154 (P < 0.05). Virus neutralization assays gave neutralizing indices of 6.6, 6.3, and 6.3 for ΔaroA M2e-CD154, ΔhtrA M2e-CD154, and ΔaroA-ΔhtrA M2e(4)-CD154 seven days post booster immunization, respectively, indicating effective neutralization of AI by serum IgG of vaccinated chickens. In a subsequent direct challenge study, specific-pathogen-free Leghorn chicks immunized with ΔaroA-ΔhtrA M2e(4)-CD154 offered significant protection against direct challenge with low pathogenic AI H7N2, but not highly pathogenic H5N1 AI. Taken together, these data suggest that these Salmonella-vectored vaccines expressing M2e in association with CD154 are effective at protecting chickens against low pathogenic AI.</abstract><cop>England</cop><pub>Poultry Science Association</pub><pmid>19834072</pmid><doi>10.3382/ps.2009-00251</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
fulltext | fulltext |
identifier | ISSN: 0032-5791 |
ispartof | Poultry science, 2009-11, Vol.88 (11), p.2244-2252 |
issn | 0032-5791 1525-3171 |
language | eng |
recordid | cdi_proquest_miscellaneous_734092145 |
source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
subjects | Animals Antigens, Viral - genetics Antigens, Viral - immunology Antigens, Viral - metabolism avian influenza Chickens Epitopes - genetics Epitopes - metabolism immune response immunoglobulin G Influenza A virus Influenza in Birds - prevention & control Influenza Vaccines - immunology liver membrane proteins microbial colonization molecular sequence data neutralization tests nucleotide sequences oral administration pathogenicity polymerase chain reaction recombinant antigens recombinant vaccines Salmonella - genetics Salmonella - metabolism Salmonella enteritidis spleen strains tonsils vaccination viral antigens Virus Shedding |
title | Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge |
url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-05T11%3A20%3A27IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Vaccination%20of%20chickens%20with%20recombinant%20Salmonella%20expressing%20M2e%20and%20CD154%20epitopes%20increases%20protection%20and%20decreases%20viral%20shedding%20after%20low%20pathogenic%20avian%20influenza%20challenge&rft.jtitle=Poultry%20science&rft.au=Layton,%20S.L&rft.date=2009-11-01&rft.volume=88&rft.issue=11&rft.spage=2244&rft.epage=2252&rft.pages=2244-2252&rft.issn=0032-5791&rft.eissn=1525-3171&rft_id=info:doi/10.3382/ps.2009-00251&rft_dat=%3Cproquest_cross%3E734092145%3C/proquest_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=734092145&rft_id=info:pmid/19834072&rfr_iscdi=true |