Research Article Avian Influenza Surveillance in the ...downloads.hindawi.com/archive/2014/965749.pdf · domestic waterfowl were purchased and were placed at each surveillance site

Research ArticleAvian Influenza Surveillance in the Danube DeltaUsing Sentinel Geese and Ducks

Alexandru Coman1 Daniel Narcis Maftei1 Razvan M Chereches1

Elena Zavrotchi1 Paul Bria1 Claudiu Dragnea1 Pamela P McKenzie2

Marissa A Valentine3 and Gregory C Gray3

1 Center for Health Policy and Public Health Institute for Social Research Faculty of PoliticalAdministrative and Communication Sciences Babes-Bolyai University 400132 Cluj-Napoca Romania

2Department of Infectious Diseases St Jude Childrenrsquos Research Hospital Center of Excellence for InfluenzaResearch and Surveillance Memphis TN 38105 USA

3 College of Public Health and Health Professions and Emerging Pathogens Institute University of Florida101 S Newell Dr Suite 2150A Gainesville FL 32610 USA

Correspondence should be addressed to Gregory C Gray gcgrayphhpufledu

Received 21 November 2013 Accepted 26 February 2014 Published 25 March 2014

Academic Editor Daniel R Perez

Copyright copy 2014 Alexandru Coman et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Highly pathogenic avian influenza (HPAI) H5N1 virus incursions from migrating birds have occurred multiple times in Romaniasince 2005 Beginning in September 2008 through April 2013 seasonal sentinel surveillance for avian influenza A viruses (AIVs)using domestic geese (Anser cygnoides) and ducks (Anas platyrhynchos) in the Danube Delta was established by placing 15 geeseand 5 ducks at seven sites Tracheal and cloacal swabs and sera collections (starting in 2009) were takenmonthlyWe studied a totalof 580 domestic birds and collected 5520 cloacal and tracheal swabs from each and 2760 sera samples All swabs were studied withreal-time reverse transcription polymerase chain reaction (rRT-PCR) for evidence of AIV Serological samples were studied withhemagglutination inhibition assays against avian H5 H7 and H9 influenza viruses From 2009 to 2013 47 swab specimens fromCot Candura Enisala and Saon screened positive for AIV further subtyping demonstrated that 14 ducks and 20 geese had cloacalevidence of H5N3 carriage Correspondingly 4 to 12 weeks after these molecular detections sentinel bird sera revealed elevated HItiters against H5 virus antigens We posit that domestic bird surveillance is an effective method to conduct AIV surveillance amongmigrating birds in delta areas

1 Introduction

Asian-lineage highly pathogenic avian influenza (HPAI)H5N1 virus has infected poultry in many countries of theworld and is thought to be highly endemic in certain deltaareas due to frequent viral incursions from large populationsof migrating birds [1 2] As of 2011 the Food and AnimalOrganization (FAO) considered HPAI H5N1 to be enzooticamong aquatic birds in Bangladesh China Egypt IndiaIndonesia and Vietnam [3]The latest report from theWorldOrganization for Animal Health revealed outbreaks of HPAIH5N1 that have occurred in Bangladesh Bhutan Cambodia

China India Korea Nepal and Vietnam in 2013 and inChina Nepal and Vietnam during early 2014 [4]

Large populations of aquatic migratory birds frequentdelta areas in Romania and evidence to date strongly suggeststhat they have introduced HPAI strains into Romaniarsquos poul-tryThe first Romanian HPAI H5N1 incursions were detectedin backyard poultry farms in October 2005 [5 6] Thevirus rapidly spread throughout the country until aggressivecontrol measures were taken the epizootic halted in July2006 [7] Laboratory studies demonstrated that the epidemicvirus was similar to H5N1 viruses previously detected inSoutheast Asia and Turkey and that migrating birds were the

Hindawi Publishing CorporationInfluenza Research and TreatmentVolume 2014 Article ID 965749 6 pageshttpdxdoiorg1011552014965749

2 Influenza Research and Treatment

LegendOutbreaks of avian flu 2005-2006Outbreak in 2007

Sentinel birds locationAgricultural areas

Artificial surfacesForest and seminatural areasWater bodiesWetlands

(km)10 5 0 10

c 1 cm = 500886 km

N

Danube Deltasentinel bird locations

Figure 1 Sentinel bird surveillance sites in the Danube Delta are represented by black rectangles Tulcea County Southeastern Romania In2011 the Periprava site was discontinued and the Letea site was added

likely source [8] A second HPAI H5N1 outbreak took placein November 2007 and was contained in Murighiol TulceaCounty Romania [9] A thirdHPAIH5N1 detection occurredamong poultry in March 2010 in six backyard poultry farmsin Letea and in one backyard poultry farm in Plauru TulceaCounty [10]These two villages are also located in theDanubeDelta As wild bird die-offs occurred in the Danube Deltabefore or in concert with these outbreaks the authors of thispaper reasoned that surveillance among migrating birds inthe Danube Delta might serve as an early warning system forfuture avian influenza virus (AIV) incursions

As HPAI viruses are thought to originate from lowpathogenic avian influenza (LPAI) viruses it is more strategicto conduct surveillance for LPAI viruses particularly amongwild birds before viruses enter domestic flocks As opposedto HPAI viruses where signs of disease occurrence are oftenmore visible due to high mortality rates LPAI often failsin causing overt signs of disease thus surveillance can bechallenging particularly among wild birds

Traditional wild bird LPAI surveillance systems ofteninvolve substantial costs for labor trapping equipment trans-portation and laboratory studies Also a number of factorscan bias results for example certain birds are easier to trapthan others and seasonality can influence the composition ofthe wild bird population hence trapping during certain time

periods can influence the overall sample diversity [11] Fur-thermore trapping of wild birds can inflict injury

In contrast monitoring domestic geese and ducks thatmix with wild birds in a sentinel system has numerousadvantages Domestic birds are quite sensitive in picking upLPAI from wild birds Surveillance costs are less as thesegeese and ducks are easier to sample and sampling can berecurrent so as to identify new LPAI introductions overtime Detections from such domestic geese and duck sentinelsystems help to identify viruses that are prone to move intodomestic birds before they become enzootic Such ldquoearlywarningrdquo detections of LPAI in the aquatic environment canhelp public health officials initiate interventions to preventthe spread of LPAI to poultry farms

With these advantages in mind we established sentineldomestic geese and duck surveillance at geographicallydiverse sites within the Danube DeltaThis report documentsearly results from this sentinel network

2 Materials and Methods

21 Location The Danube Delta is the second largest riverdelta in Europe after the Volga Delta The total delta area isapproximately 4125 km2 of which approximately 85 is situ-ated in Tulcea County Romania In 2008 we initiated active

Influenza Research and Treatment 3

sentinel domestic geese (Anser cygnoides) and duck (Anasplatyrhynchos) surveillance in the Danube Delta surveillancewas first initiated in one location Cot Candura and thenexpanded to seven sites by August 2009 (Figure 1)

In an effort to use representative sites for sentinelbird surveillance researchers selected sites that were geo-graphically diverse while also taking into account thegeographic density of wild bird populations in differentareas of the Danube Delta Specific locations near wet-lands or lakes were targeted where previous AIV had beenisolated from dead waterfowl or where primary reservoirspecies frequently gather [12] In the surveillance sitesshelters and open fenced enclosures (Figures 2 and 3)were constructed to hold 20 domestic birds The surveil-lance sites were located at (1) Saon (N45∘ 131015840 010158401015840 E28∘331015840 010158401015840) (2) Ceamurlia de Jos (N440441015840910158401015840 E2804310158402610158401015840)(3) Periprava (N4502110158400510158401015840 E2903710158404410158401015840) replaced in 2011by Letea (N4501010158400510158401015840 E2900010158404910158401015840) (4) Cot Candura(N450141015840 5210158401015840 E2900410158404410158401015840) (5) Caraorman (N4500410158402810158401015840E2902410158405510158401015840) (6) Murighiol (N4500010158405110158401015840 E2900710158403710158401015840) and(7) Enisala (N4405210158405510158401015840 E2804810158405910158401015840) (Figure 1)

22 Bird Selection and Sampling Surveillance was initiated in2008 and is still in progress at the time of this report (February2014) Each year in early September 140 young free rangingdomestic waterfowl were purchased and 20 were placed ateach surveillance site (sim15 geese and 5 ducks) Sentinel geeseand duck placement during this time period coincides withwild bird migration into the Danube Delta Both geese andducks were used to increase the probability of contact withmigrating wild geese and duck species Geese were chosensince they tend to lead the flock back to the pens in theevening after a day spent on the lakesThe numbers of malesto females (1 4) were chosen in order to avoid fighting amongmales Beginning in the third year of the surveillance sentinelbirds were banded such that the identity and health of eachbird could be tracked over the influenza season Prior to birdbanding in order to ensure the naivety of the flock any flockthat had a positive swab sample was removed and replacedwith verified healthy birdsThis was accomplished via testingbirds for influenza A virus using real-time RT-PCR (rRT-PCR) and serology to ensure that they were naıve to influenzaA viruses prior to release at sentinel sites

The sentinel birds were free to move around in localwaters and to mix with wild waterfowl Each evening thesentinel birdswere encouragedwith food to return to the pro-tective enclosure Nearly every month cloacal and trachealswabs were collected by a veterinary team which traveled tothe sentinel sites via boat or car

The veterinary team also collected blood samples from arandomselection of half of the sentinel birds in order to deter-mine their serologic status Birds that were lost seropositiveor rRT-PCR positive for influenza were replaced The birdswere tended by local villagersfishermen who agreed to assistin the surveillance study

23 Ethical Statement Actions were taken to ensure animalhealth and well-being throughout the study via enlistinglicensed public health veterinarians from the Tulcea County

Figure 2 Photograph of a typical sentinel bird shelter Caraormansite Danube Delta September 2010

Figure 3 Photograph of a sentinel bird surveillance site DanubeDelta October 2013

Health Department to collect sera and swabs from thesentinel birds Additionally the study was approved by theInstitutional Animal Care and Use Committee (IACUC) atthe University of Florida

24 Laboratory Work Tests were conducted in accordancewith the Diagnostic Manual for Avian Influenza of theEuropeanUnion [13] and theOIEManual of Diagnostic TestsandVaccines for Terrestrial Animals (WorldOrganization forAnimal Health 2011) RNA was extracted from swabs usingviral RNA kits (Qiagen Alameda CA USA) and analyzedby rRT-PCR targeting the influenza A virus matrix gene Allpositive samples were sent to the National Reference Labo-ratory for Avian Influenza Bucharest Romania followed bytheWeybridge EUOIEFAO reference laboratory for furtherinvestigation (virus isolation sequencing and genotyping)Positive samples were also sent to the Global PathogensLaboratory at the University of Florida for molecular studyThe detection of new AIV strains was quickly communicatedto the local and central veterinary authority

25 Serological Assays FromSeptember 2009 throughMarch2013 serological samples were obtained from a random


Table 1 Number and location of real-time RT-PCR influenza A detections among sentinel birds in Romaniarsquos Danube Delta 2009ndash2013

Date Collection site Type of sample Number of positive samplestotalnumber of tested samplesyear ()

012009 Cot Candura Cloacal swabs 10400 (25)092010 Cot Candura Cloacal swabstracheal swabs 20400 (5)102011 Enisala Cloacal swabs 4400 (1)032013 Saon Cloacal swabstracheal swabs 9400 (225)032013 Cot Candura Cloacal swabstracheal swabs 4400 (1)

Table 2 Geese or duck sera with elevated serum hemagglutinationinhibition assays against H5 H7 and H9 hemagglutinin antigensCollection period 2010ndash2013

Collection site Hemagglutinin inhibition assay ( elevated)lowast

H5 () H7 () H9 ()Ceamurlia de Jos 1 32 (1)Cot Candura 1 256 (77) 1 128 (13)Enisala 1 16 (3)Letea 1 16 (25) 1 64 (1)Saon 1 16 (4) 1 16 (1)Caraorman 1 128 (05)lowastA titer ge1 16 was considered positive total number of sera samplescollected = 300collection site

selection of half of the sentinel birds at each location FromSeptember 2009 through September 2011 serological sampleswere characterized using an ELISA blocking kit for AIVtype A antibody detection (Pourquier Institute now partof Idexx Laboratories Inc Westbrook ME USA) Positivesera were then tested using a hemagglutination inhibition(HI) assay Beginning in September 2011 only the HI testwas performed All serological testing was conducted inthe Tulcea County Sanitary Veterinary and Food SafetyLaboratoryH5N2H7N3 andH9N2 avian influenza antigens(Istituto Zooprofilattico Sperimentale delle Venezie LegnaroItaly) were used in the HI assays The study used theseantigens for screening since H5 H7 and H9 influenza strainshave caused AIV outbreaks in humans and in birds

3 Results

Cloacal and tracheal swabs collected from each sentinel bird(5520 each) and sera samples collected from a subset ofsentinel birds (2760) between September 2008 and April2013 and September 2009 and April 2013 respectively werescreened for evidence of AIV infection From 2009 through2013 47 swabs from 47 birds at 3 distinct locations screenedpositive for influenza A virus on five different occasions(Table 1) After subtyping the virus results revealed 34 cloacalswabs positive for H5N3 H5N3 virus was present in 14 ducksand 20 geese although no clinical signs were observed insentinel or wild birds from the sentinel sites The remaining13 influenza A rRT-PCR positive samples from Enisala andSaon were confirmed by the Romanian National ReferenceLaboratory and the Global Pathogens Laboratory at theUniversity of Florida respectively however no virus was

isolated or sequenced from the remaining 13 samples due tolow viral load H5N3 detected in 2009 at Cot Candura wasthe first LPAI virus ever isolated in Romania Each new AIVstrain detected was quickly communicated to both the localand national veterinary authorities

In the subset of previously immunologically naıve sen-tinel birds serological assays revealed elevated HI titersagainst H5 H7 and H9 viruses (Table 2) We found elevatedHI antibodies against the H5N2 antigen within 4ndash12 weeksafter the first molecular detections of H5N3 virus Approxi-mately 8 weeks after the HI elevations antibody titers wanedto levels lt1 16

4 Discussion

National AIV surveillance programs within the EuropeanUnion vary in terms of the resources and the types of surveil-lance methods used [14] Four main surveillance strategiesare employed (1) active surveillance involving testing of livecaught wild birds (2) active surveillance of hunted birds(3) active surveillance (periodic sampling) using sentineldomestic birds kept in high-risk areas and (4) passive surveil-lance and laboratory study of dead wild birds or poultrywhen unusual mortality is detected [15] Active surveillanceinvolving wild caught birds proves suitable for detectingboth HPAI and LPAI viruses in high-risk areas while activesurveillance of hunted birds has frequently been successfulin detecting LPAI viruses Use of the hunted bird approachhowever does not permit sampling for the entire respiratoryseason Active surveillance of sentinel domestic birds is usedinfrequently but has been useful in both HPAI and LPAIvirus detections Romania relies upon the passive surveillancestrategy among domestic and wild birds and hence AIVsurveillance is sparse and dependent upon the discovery andstudy of sick or dead wild birds For comparison purposesthe Romanian passive system of surveillance did not uncoverany evidence of LPAI viruses during the study periodWhen analyzing financial costs use of methods 1 and 2 addadditional financial constraints due to the requirement ofadditional traps and net cannons prolonged time spent in thefield as well as the need for additional boats and gas Theseincurred costs would supplement those already incurred bythe sentinel surveillance method

Among the sentinel geese and ducks included in thecurrent surveillance study we detected several LPAI H5N3viruses and found serological evidence that LPAI H7 andH9 virus strains were also circulating (Table 2) The detec-tion of elevated and rapidly reduced serologic titers against


LPAI virus suggests that these infections may be subclinicaltransient and missed by periodic tracheal and cloacal swabsfollowed by molecular assays alone

Detection of LPAI in sentinel bird populations serves asan important precursor for preventingHPAI outbreaks Priorto HPAI outbreaks circulating AIVs undergo mutationsand can transform into LPAI oftentimes without causingovert mortality in flocks until it mutates into a HPAI Thepublic health response to both LPAI and HPAI remains thesame this includes culling infected birds In LPAI situationsthis prevents the spread of LPAI and contact with existingviruses in animals which could result inmutation and hencepotential emergence of HPAI Thus surveillance for LPAIin bird populations remains a key component in helping toprevent HPAI outbreaks

During the study period only 2 documented H5N1outbreaks occurred within birds in the Danube Deltamdashone in Letea and one in Plauru The outbreaks occurredin 2010 within 2 weeks of each other We posit that thesentinel surveillance did not detectH5N1 incursions since thedistance between the closest sentinel site and the outbreakarea was 15 km In 2011 in order to enhance study detectionthe Periprava site was discontinued and a new sentinel site inLetea was established

5 Conclusion

We propose that domestic geese and ducks can be used asan effective AIV sentinel surveillance system especially indelta areas where the domestic birds can freely mix withlarge populations of migrating birds Using this method wewere able to detect the presence of H5N3 via molecularsurveillance as well as find serological evidence of circulatingH7 and H9 viruses Additionally for our sentinel birdsurveillance we estimated that costs were less expensivethan using other AIV surveillance methods such as trappinghunting or collecting wild bird carcasses Use of sentinelbird surveillance may also provide a more representativeexample of circulating viruses due to the species biases thataccompanies hunting or sampling from deceased birdsThusdomestic bird surveillance is less invasive less expensive andmore effective than using other surveillance strategies

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thiswork was funded by theNational Institute of Allergy andInfectious Diseases the National Institutes of Health underContract no HHSN266200700005C and multiple Grantslisted below R01 AI068803-ARRA supplement (Dr Gray)from theNational Institute of Allergy and Infectious Diseasesand multiple grants (Dr Gray) from the US Department ofDefense Armed Forces Health Surveillance Centerrsquos GlobalEmerging Infections Surveillance and Response Program

The study was approved by the University of Florida AnimalCare and Use CommitteeThe authors thank Professor DavidA Halvorson DVM of the College Of Veterinary Medicineat the University of Minnesota for his early advice in settingup the sentinel bird surveillance They also thank theircolleagues fromTulcea County Sanitary Veterinary and FoodSafety Authority for their collaboration

References

[1] M P Ward D N Maftei C L Apostu and A R SuruldquoAssociation between outbreaks of highly pathogenic avianinfluenza subtypeH5N1 andmigratory waterfowl (family anati-dae) populationsrdquo Zoonoses and Public Health vol 56 no 1 pp1ndash9 2009

[2] M P Ward D Maftei C Apostu and A Suru ldquoEnvironmentaland anthropogenic risk factors for highly pathogenic avianinfluenza subtype H5N1 outbreaks in Romania 2005-2006rdquoVeterinary ResearchCommunications vol 32 no 8 pp 627ndash6342008

[3] Approaches to Controlling Preventing and Eliminating H5N1Highly Pathogenic Avian Influenza in Endemic Countries Ani-mal Production and Health Paper Food and Agriculture Orga-nization Rome Italy 2011

[4] Update on Highly Pathogenic Avian Influenza in Animals (TypeH5 and H7) World Organisation for Animal Health 2014

[5] A Nicoll ldquoAvian influenza detected in Turkey and RomaniardquoEuro Surveillance vol 10 no 10 p E0510131 2005

[6] G Cattoli A Fusaro I Monne and I Capua ldquoH5N1 virusevolution in Europemdashan updated overviewrdquo Viruses vol 1 pp1351ndash1363 2009

[7] F a A O o t U Nations Fewer Bird Flu Outbreaks This Year-Sign of Progress FAO Says Food and Agriculture Organizationof the United Nations 2007

[8] G Oprisan H Coste E Lupulescu et al ldquoMolecular analysis ofthe first avian influenza H5N1 isolates from fowl in RomaniardquoRoumanian Archives of Microbiology and Immunology vol 65no 3-4 pp 79ndash82 2006

[9] A Nagy V Vostinakova Z Pindova et al ldquoMolecular andphylogenetic analysis of the H5N1 avian influenza virus causedthe first highly pathogenic avian influenza outbreak in poultryin theCzechRepublic in 2007rdquoVeterinaryMicrobiology vol 133no 3 pp 257ndash263 2009

[10] S M Reid W M Shell G Barboi et al ldquoFirst reported incur-sion of highly pathogenic notifiable avian influenza A H5N1viruses from clade 232 into European poultryrdquo Transboundaryand Emerging Diseases vol 58 no 1 pp 76ndash78 2011

[11] A Globig A Baumer S Revilla-Fernandez et al ldquoDucks assentinels for avian influenza in wild birdsrdquo Emerging InfectiousDiseases vol 15 no 10 pp 1633ndash1636 2009

[12] I Iglesias A M Perez A De la Torre M J Munoz MMartınez and J M Sanchez-Vizcaıno ldquoIdentifying areas forinfectious animal disease surveillance in the absence of pop-ulation data highly pathogenic avian influenza in wild birdpopulations of Europerdquo Preventive Veterinary Medicine vol 96no 1-2 pp 1ndash8 2010

[13] ldquo2006437EC Commission Decision of 4 August 2006 approv-ing a diagnostic manual for avian influenza as provided for inCouncil Directive 200594ECrdquoOfficial Journal of the EuropeanUnion Legislation vol 237 pp 1ndash27 2006


[14] A C Breed K Harris U Hesterberg et al ldquoSurveillance foravian influenza in wild birds in the European Union in 2007rdquoAvian Diseases vol 54 no 1 pp 399ndash404 2010

[15] U Hesterberg K Harris D Stroud et al ldquoAvian influenzasurveillance in wild birds in the European Union in 2006rdquoInfluenza and other Respiratory Viruses vol 3 no 1 pp 1ndash142009

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LegendOutbreaks of avian flu 2005-2006Outbreak in 2007

Sentinel birds locationAgricultural areas

Artificial surfacesForest and seminatural areasWater bodiesWetlands

(km)10 5 0 10

c 1 cm = 500886 km

N

Danube Deltasentinel bird locations

Figure 1 Sentinel bird surveillance sites in the Danube Delta are represented by black rectangles Tulcea County Southeastern Romania In2011 the Periprava site was discontinued and the Letea site was added

likely source [8] A second HPAI H5N1 outbreak took placein November 2007 and was contained in Murighiol TulceaCounty Romania [9] A thirdHPAIH5N1 detection occurredamong poultry in March 2010 in six backyard poultry farmsin Letea and in one backyard poultry farm in Plauru TulceaCounty [10]These two villages are also located in theDanubeDelta As wild bird die-offs occurred in the Danube Deltabefore or in concert with these outbreaks the authors of thispaper reasoned that surveillance among migrating birds inthe Danube Delta might serve as an early warning system forfuture avian influenza virus (AIV) incursions

As HPAI viruses are thought to originate from lowpathogenic avian influenza (LPAI) viruses it is more strategicto conduct surveillance for LPAI viruses particularly amongwild birds before viruses enter domestic flocks As opposedto HPAI viruses where signs of disease occurrence are oftenmore visible due to high mortality rates LPAI often failsin causing overt signs of disease thus surveillance can bechallenging particularly among wild birds

Traditional wild bird LPAI surveillance systems ofteninvolve substantial costs for labor trapping equipment trans-portation and laboratory studies Also a number of factorscan bias results for example certain birds are easier to trapthan others and seasonality can influence the composition ofthe wild bird population hence trapping during certain time

periods can influence the overall sample diversity [11] Fur-thermore trapping of wild birds can inflict injury

In contrast monitoring domestic geese and ducks thatmix with wild birds in a sentinel system has numerousadvantages Domestic birds are quite sensitive in picking upLPAI from wild birds Surveillance costs are less as thesegeese and ducks are easier to sample and sampling can berecurrent so as to identify new LPAI introductions overtime Detections from such domestic geese and duck sentinelsystems help to identify viruses that are prone to move intodomestic birds before they become enzootic Such ldquoearlywarningrdquo detections of LPAI in the aquatic environment canhelp public health officials initiate interventions to preventthe spread of LPAI to poultry farms

With these advantages in mind we established sentineldomestic geese and duck surveillance at geographicallydiverse sites within the Danube DeltaThis report documentsearly results from this sentinel network

2 Materials and Methods

21 Location The Danube Delta is the second largest riverdelta in Europe after the Volga Delta The total delta area isapproximately 4125 km2 of which approximately 85 is situ-ated in Tulcea County Romania In 2008 we initiated active





















3 Results




4 Discussion







5 Conclusion




Acknowledgments



References






















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Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































3 Results




4 Discussion







5 Conclusion




Acknowledgments



References






















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Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results




4 Discussion







5 Conclusion




Acknowledgments



References






















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Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















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Acknowledgments



References






















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Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























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Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Avian Influenza Surveillance in the ...downloads.hindawi.com/archive/2014/965749.pdf · domestic waterfowl were purchased and were placed at each surveillance site