research article molecular characterization of chicken...

Research ArticleMolecular Characterization of Chicken Anemia VirusCirculating in Chicken Flocks in Egypt

Mohammed AboElkhair12 Alaa G Abd El-Razak3 and Abd Elnaby Y Metwally4

1 Department of Virology Faculty of Veterinary Medicine University of Sadat City Sadat City 32897 Minoufiya Egypt2 Research Center for Animal Hygiene and Food Safety Obihiro University of Agriculture and Veterinary Medicine Inada 2-11Hokkaido Obihiro 080-8555 Japan

3Department of Bird and Rabbits Medicine Faculty of Veterinary Medicine University of Sadat CitySadat City 32897 Minoufiya Egypt

4Animal Health Research Institute Kafr El Sheikh Provincial Laboratory Kafr El Sheikh Egypt

Correspondence should be addressed to Mohammed AboElkhair maboelkhair2004yahoocom

Received 23 June 2014 Revised 1 September 2014 Accepted 4 September 2014 Published 15 September 2014

Academic Editor Finn S Pedersen

Copyright copy 2014 Mohammed AboElkhair et alThis is an open access article distributed under the Creative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Introduction Although many previous studies reported detection of chicken anemia virus (CAV) in Egypt since 1990 genomiccharacterization of this circulating CAV has not been published In the present study four nucleotide sequences of detected CAVwere genetically characterizedMethodsThese nucleotide sequences were obtained from commercial chicken flocks in two differentlocations of Egypt during 2010The target region for sequencing was 675 bp nucleotide of partial coding region of VP1 proteinThenucleotide and deduced amino acid sequences of the detectedCAVwere aligned and compared toworldwideCAV isolates includingcommonly used vaccine strains Phylogenetic analysis of these sequences was also carried out Results Our results showed that allthe Egyptian CAV sequences were grouped in one group with viruses from diverse geographic regions This group is characterizedby amino acids profile 75I 97L 139Q and 144Q in VP1 The phylogenetic and amino acid analyses of deduced amino acid indicatedthat the detected CAV sequences differ from CAV vaccine strains Conclusion This is the first report that describes molecularcharacterization of circulating CAV in Egypt The study showed that the detected CAV in Egypt are field viruses and unrelated tovaccine strains

1 Introduction

Chicken anemia virus (CAV) is an economically importantpathogen with a worldwide distribution CAV is a small DNAvirus with a closed circular negative single stranded DNAgenome It belongs to genus Gyrovirus of family Circoviridae[1] The genome consists of three partially overlapping openreading frames encoding three viral proteins VP1 (516 kDa)the major viral capsid protein VP2 (24 kDa) a novel dualspecificity protein phosphatase [2] that also probably actsas scaffolding protein during virion assembly [3] and VP3(136 kDa) also called apoptin which has been shown to haveapoptotic activity in transformed cell lines [4] VP1 shows thehighest nucleotide variability therefore it is usually used forgenetic characterization and molecular studies of CAV [5 6]

InfectionwithCAV constitutes a serious economic threatespecially to the broiler industry and the producers of specific

pathogen free (SPF) eggs The clinical signs are mainlynoticed in young chicks of 10ndash14 days of age which acquirethe infection vertically Chickens older than 2-3 weeks of ageare also susceptible to infection but only develop a subclinicaldisease evidenced by poor vaccine response [7] The diseaseis characterized by aplastic anemia and generalized lymphoidatrophy with concomitant immunosuppression and frequentassociationwith secondary viral bacterial parasitic or fungalinfections [7] Mortalities and morbidities due to CAVinfection may reach 55 and 80 respectively [8]

In Egypt many previous publications have reporteddetection of CAV in chicken population (cited in [9ndash11])however genomic characterization from these isolates hasnot been published The aim of this study was to identifyCAV obtained from farms with problems associated withimmunosuppression and to determine their relationship withvaccine and other reference strains

Hindawi Publishing CorporationAdvances in VirologyVolume 2014 Article ID 797151 6 pageshttpdxdoiorg1011552014797151

2 Advances in Virology

Table 1 Samples collected for the current study

Province Numbers of the flocks Type of samples Age of the flockSharkia Three Tissue homogenates 12ndash35 daysKafr El Sheikh One Tissue homogenate 25 daysMinoufiya One Liver 30 days

2 Materials and Methods

21 Samples Collection Tissue homogenates were collectedfrom Sharkia andKafr El Sheikh provincesThe homogenatescontained thymus loops bone marrow bursa of Fabriciusliver spleen and intestines samples Tissue homogenatesof Sharkia province were collected from three commercialbroiler flocks The flocks ranged in age from 12 to 35 daysrepresenting different breeds and localities in the provinceTissue homogenate of Kafr El Sheikh Province was onlycollected from one commercial broiler flock aged 25 daysFive liver samples were also collected from a commercialbroiler chicken flock aged 30 days from Minoufiya provinceAll flocks of collected samples were showing clinical signsand lesions indicative of CAV infection [10] The placesnumbers of flocks and age of flocks of selected samples arelisted in Table 1 All samples were stored frozen at minus70∘C forsubsequent DNA extraction

22 Sample Preparation Tissues were homogenized in amortar with sterile sand and phosphate-buffered saline Celldebris and sand were eliminated by centrifugation and thesupernatants were collected and stored at minus70∘C

23 DNA Extraction and PCR Amplification DNA wasextracted from the supernatant of liver and tissuehomogenates by QIAamp DNA mini kit (Qiagen IncValencia CA) according to the manufacturer instructionsThe oligonucleotide primers 51015840-GAC TGT AAG ATG GCAAGA CGA GCT C-31015840 and 51015840-GGC TGA AGG ATC CCTCAT TC-31015840 were used to amplify a 675 bp DNA fragmentof Vp1 [12] from nucleotides 823 to 1498 numbering iscorresponding to the Del-Ros strain (GenBank AF313470)The PCR assay was performed in a final volume of 50 120583LReddy-Mix 18120583L PCR grade water 1 120583L of each primer and5 120583L template The amplification was performed under thefollowing conditions one cycle of initial denaturation stepat 95∘C for 15min followed by 30 cycles of 95∘C for 1min56∘C for 1min and 72∘C for 1min representing denaturationannealing and extension steps respectively and finally onecycle of final extension step at 72∘C for 5min The amplifiedproducts were analyzed using electrophoresis unit Theywere loaded to 1 agarose stained by ethidium bromidevisualized under 304 nm ultraviolet (UV TransilluminatorMajor Science) and photographed by a gel documentationsystem using Canon power-shot camera

24 Nucleotide Sequence Analysis Sequencing was per-formed in both directions with virus specific primersSequences were analyzed using BioEdit program [13] This

program was also used to read the sequencing electrophero-grams to exclude nucleotide ambiguity The phylogeneticanalysis was based on the deduced amino acids of 579nucleotides encompassing hypervariable region of vp1 Thedetected sequences were compared with others deposited inGenBank by multiple alignment with the Clustal W includedin Bioedit software Phylogenetic relationships were evalu-ated by the Neighbor Joining method present in the MEGAversion 6 software [14] with 1000 bootstrap replicationsSequence data were submitted to GenBank with accessionnumbers KJ955377 toKJ955380 for the Egy-1 CAV to the Egy-4 CAV respectively

25 Attempted Isolation of the Virus Isolation of CAV fromthe tissue homogenate of the PCR-positive samples wasattempted in embryonated SPF eggs through yolk sac inoc-ulation [7] Tissue samples were prepared according to [15]Briefly the homogenate was mixed with an equal volume ofchloroform for 15min in a shaker Three times of repeatedfreezing and thawing were applied and then the homogenatewas centrifuged for 20min at 3000 rpm The supernatantswere used for SPF egg inoculation Each homogenate wasinjected in 5 SPF eggs (100 120583Legg) After 14 days all embryostissues were homogenized DNA extraction and PCR ampli-fication were carried out from the supernatant of these tissuehomogenates in the same condition as described above Thewhole isolation attempt was repeated once

3 Results

31 Samples and Clinical Signs All affected birds were atage of 12 to 35 days All affected flocks showed signs ofanemia generalized weakness depression pale comb andwattles growth retardation and high mortality rates Thepostmortem examination revealed pale and enlarged liverand spleen mild to severe thymus atrophy and atrophiedbursa of Fabricius

32 PCR Amplification Analysis of PCR amplification ofthe extracted DNA from tissue samples by agarose gelelectrophoresis indicated DNA bands of corrected size asexpected with a length of 675 bp The authenticity of PCRamplification was confirmed by the nucleotide sequencingAll of the liver samples from the flock in Minoufiya provinceshowed negative results Also all SPF embryo homogenateswere PCR negative

33 Nucleotide Sequence Analysis The four nucleotidesequences of detected CAV displayed a limited diversityTotal nucleotide variation among the sequences ranged from

Advances in Virology 3

Table 2 Nucleotide and deduced amino acid difference and identitymatrices among Egyptian CAV sequences

(a) Nucleotide sequence difference count matrix

Egy-1 CAV Egy-2 CAV Egy-3 CAV Egy-4 CAVEgy-1 CAV IDEgy-2 CAV 19 IDEgy-3 CAV 29 22 IDEgy-4 CAV 1 18 28 ID

(b) Amino acid sequence identity matrix


1 to 29 nucleotides (Table 2(a)) They showed 949ndash998similarity between them Egy-3 CAV sequence showed thelowest similarity 949ndash962 with the other CAV sequencesEgy-1 CAV and Egy-4 CAV showed the highest similarity998 Compared to isolates from other geographical placesaround the world two Egyptian CAV sequences (Egy-1 CAVand Egy-4 CAV) were found to have maximum homologywith an Argentinean isolate Arg0021-3 by 99 and 100respectively whereas the Egy-2 CAV showed 98 homologywith a Cameroon strain CMR09-731 and the Egy-3 CAVwas 99 similar to a Cameroon strain CMR09-485

34 Amino Acids Sequence Analysis The four detected CAVsequences showed 989 to 100 identity with each other atthe level of amino acid sequence (Table 2 (b))Themaximumdifference was between Egy-3 CAV and Egy-1 CAV Thesetwo sequences (Egy-3 CAV and Egy-1 CAV) varied onlyin two amino acids The deduced amino acid sequencesof the Egyptian CAV sequences and some vaccine strainswere aligned The analysis was carried out to outline theshared amino acid residues between detected viruses andsome vaccine strains (Cux-1 Del-Ros and 26PA) (Table 3)Also other strains from all over the world were includedThe Egyptian CAV sequences showed consensus amino acidsequence at positions 75 97 139 and 144 of VP1 At position22 however Egy-1 CAV Egy-2 CAV and Egy-4 CAV hadhistidine (H) but Egy-3 CAV had asparagine (N) instead ofH As shown in Table 3 all Egyptian CAV sequences showedsequence differencewith regard to vaccine strains At position75 the Egyptian CAV had isoleucine (I) instead of valine (V)at position 97 they had leucine (L) instead ofmethionine (M)at position 139 they had glutamine (Q) instead of lysine (K)and at position 144 they also had Q instead of glutamic acid(E) N or aspartic acid (D)

35 Phylogenetic Analysis The deduced amino acid sequen-ces of the partial vp1 sequence of Egyptian CAV werecompared with those of other sequences deposited in theGenBank All detected CAVs in Egypt were grouped together

in one group (Figure 1) The topology of the phylogenetictree revealed the presence of three groups that were definedin this study as I II and III The tree topology alsoshowed that the Egyptian sequences were related to dif-ferent sequences such as NIE1904118Nigeria CMR09-731andCMR09-485Cameroon CL37Chile CAV-BIndia AN-China 23China C1A-1USA Arg0021-3Argentina and BD-3Bangladesh which were classified as group II according to[16] However different commonly used vaccine strains forexample Nobilis P4 Del-Ros 26PA andCux-1 groupedwithother sequences in groups I and III (Figure 1)

4 Discussion

It was reported that CAV spread among chicken in Egyptsince the early 1980s when several outbreaks occurred inmany breeds [9] The presence of CAV was confirmed bydetection of both CAV antibodies and genome in both meatand egg type chicken flocks [9ndash11] However the molecularcharacterization from these viruses has not been publishedCurrently it is so important to characterize circulating CAVscirculating in Egypt to improve methods of virus control andto determine the relationship of circulating CAVwith vaccinestrains and other CAV strains Particularly a large number ofisolates have been fully or partially sequenced These isolateswere obtained in many countries for example BangladeshBrazil China Malaysia Slovenia the United States [1] andArgentina [6] Also a number of CAV sequences have beenreported in African continent for example Nigeria [5]Central African Republic and Cameroon [17] and morerecently from South Africa [18] Comparison of all sequencedata indicates that these isolates can be divided into 3 or 4distinct groups [1] Vp1 gene sequence is commonly used todetermine the relationship of different CAV isolates due tothe fact that most of the amino acid substitutions betweenisolates lie in vp1 gene andmore specifically in theN-terminalhalf of vp1 gene [6 19] Moreover a hypervariable regionspanning from amino acid positions 139 to 151 in Vp1 wasidentified [20] Islam et al [16] found that five of 16 commonlyvariable amino acid positions of the whole vp1 fall withinthis small region Therefore in the present study we usedpartial sequencing of vp1 including hypervariable region asa tool to study the molecular characterization of EgyptianCAV sequences Although the Egyptian detected CAVsshowed up to 51 difference at the nucleotide sequencelevel (Table 2(b)) they are almost identical at the aminoacid level This indicated that they differed only by silentmutations

According to a previous study [16] CAV isolates can begrouped into three different groups based on the amino acidresidues at positions of 75 97 139 and 144 of amino acidsequence of VP1 protein In the current study all detectedCAV sequences showed the same amino acid profiles 75I97L 139Q and 144Q Furthermore the phylogenetic analysisindicated that all detected Egyptian CAV sequences belong togroup II based on Islam et al classification [16]Themembersof this group are from very diverse geographic origins Also


Table 3 Amino substitution in VP1 sequence of CAV isolates

Isolate Amino acid positions22 48 75 83 97 125 139 141 144 157

Consensus H A V I M I K Q Q VEgy-1 CAVEgypt sdot sdot I sdot L sdot Q sdot sdot sdot

Egy-2 CAVEgypt sdot sdot I sdot L sdot Q sdot sdot sdot

Egy-3 CAVEgypt N sdot I sdot L sdot Q sdot sdot sdot


Cux-1 M Germany sdot sdot sdot sdot sdot sdot sdot sdot D sdot

Cux-1 N Germany sdot sdot sdot sdot sdot sdot sdot sdot D sdot

Del-RosUSA sdot sdot sdot sdot sdot sdot sdot sdot N sdot

ConnBUSA sdot sdot sdot sdot sdot sdot sdot sdot E sdot

26PAUSA sdot sdot sdot sdot sdot sdot sdot sdot E MSMSC-1Malaysia sdot sdot I sdot L sdot sdot sdot sdot sdot

CIA-1USA N sdot I sdot L sdot Q sdot sdot sdot

A2Japan sdot sdot sdot sdot sdot sdot sdot E E MAF448446China sdot sdot sdot sdot sdot sdot sdot E sdot

BD-3Bangladesh sdot sdot I sdot L sdot Q sdot sdot sdot

NIE1904118Nigeria sdot sdot I sdot L sdot Q sdot sdot sdot

Isolate704Australia sdot sdot I sdot L sdot Q sdot sdot sdot

ArgA0021-3Argentina sdot sdot I sdot L sdot Q sdot sdot sdot

AN-China 23China N sdot I sdot L sdot Q sdot sdot sdot

CMR09-731Cameroon sdot sdot I sdot L sdot Q sdot sdot sdot

CL37Chile sdot sdot I sdot L sdot Q sdot sdot sdot

most of strains isolated from African continent are includedin this group

Interestingly Egy-3 CAV has a unique amino acid sub-stitution at position 22 which is similar to CIA-1 and AN-China 23 isolates It had N instead of H at this position vanSanten et al [21] considered this residue to be important fordistinguishing of CAV strains

It was suggested that the genetic grouping of CAV strainsmight be related to different biological properties of thesestrains Renshaw et al [20] suggested that 139Qand 144Qcouldaffect the rate of replication or spread of infection in culturedcellsThey have proved that presence of these two amino acidsis associated with decreased rate of spread in cell cultureBased on this finding all detected Egyptian CAVs could havea reduced ability to spread in cell culture

Also all detected Egyptian CAVs had threonine inposition 89 instead of alanine It was suggested that thissubstitution is associated with attenuation [5] Others [22]however suggested that the previous mutation should becombined with 75I 125L 141L and 144E to produce attenua-tion No one of Egyptian detected CAV sequences displayedthis combination

Although the evidence of use of live attenuated CAV vac-cines occurring in the Egyptian poultry industry is not readilyavailable the topology of the phylogenetic tree showed thatthe CAVEgyptian sequences are in a distant relationshipwithdifferent vaccine strains commonly used for exampleNobilisP4 Del-Ros and Cux-1 All Egyptian CAV sequences are

included in group II whereas vaccine strains are located ingroups I and III

Failure of isolation of CAV in SPF eggs may be due tothe low virus concentration in the inoculum or due to thepresence of CAV antibodies in the inoculated SPF eggs Itwas reported that SPF eggs might contain CAV antibodies[1] Also the failure of detection of CAV DNA in livers ofMinoufiya chicken flock could be due to inability of the usedprimers to amplify the proposed CAV because of mutationswithin the primer location as we used in this study only onepair of primer set It was proposed that mutation in locationof the primer could lead to loss of detection of some CAVstrains [5]

There are some CAV sequences from Egypt (unpublishedreport) available in the GenBank with accession numbersHM4608791 to HM4608831 However these sequencesshared only around 124 nucleotides of VP1with our sequenceSo the alignment with our sequences was difficult

5 Conclusion

This is the first report that describes the molecular char-acterization of circulating CAV in Egyptian poultry farmsAlthough the number of samples was limited our phyloge-netic analysis revealed that CAV strains detected in Egyptwere in one group that showed similarity to several strainsall over the world particularly to those circulating in Africanand South American continentsThe results presented in this


AN-China23ChinaHQ8720301

CMR09-485CameroonHE6869901

Egy-3 CAVEgyptKJ955379

CIA-1USAL147671


130SloveniaDQ016138

704AustraliaU654141

NIE1904118NigeriaAJ888524

CL37ChileJQ3082131


CAV-BIndiaAY583756

G6JapanAB1194481

SMSC-1MalaysiaAF2858821

BD-3BangladeshAF395114

ArgA0021-3ArgentinaEU8717831



TR20JapanAB0274701

Conn-BUSAU695481

82-2JapanD319651

CAV-AIndiaAY583755

AH9410JapanAB0465901

Cux-1MGermanyM812231

Cux-1NGermanyM559181

Del-RosUSAAF313470

ChinaAF448446

CAU2697AustraliaAF2279821

PallisterAustraliaS714881

BL-5MalaysiaAF5270371

98D02152USAAF311890

Nobilis P4 VaccineAJ890284

26P4USAD100681

A2JapanAB0312961

75

51

64

97

0005

I

III

II

Figure 1 Phylogenetic relationship among 33 different CAV isolates based on partial amino acid sequences of VP1The tree was constructedby the Neighbor Joining algorithm Relevant nodes with significant bootstrap values (gt50) over 1000 replicates are indicated Egyptiandetected CAVs are marked in black triangles The horizontal lines indicate the relative nucleotide distance between samples

study clearly showed that the detected CAV sequences belongto field viruses and are not vaccine strains

Conflict of Interests

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

Acknowledgments

The authors would like to thank Dr Nazim AA for hisassistance in sample collection Also they thank Trinuh QD

Obihiro University of Agricultural and Veterinary MedicineJapan for his assistance and El-Naggar RF for her technicalassistance

References

[1] K A Schat ldquoChicken anemia virusrdquo Current Topics in Microbi-ology and Immunology vol 331 pp 151ndash183 2009

[2] M A Peters D C Jackson B S Crabb and G F BrowningldquoChicken anemia virus VP2 is a novel dual specificity proteinphosphataserdquo Journal of Biological Chemistry vol 277 no 42pp 39566ndash39573 2002


[3] MHM Noteborn C A J Verschueren G Koch andA J VanDer Eb ldquoSimultaneous expression of recombinant baculovirus-encoded chicken anaemia virus (CAV) proteins VP1 and VP2is required for formation of the CAV-specific neutralizingepitoperdquo Journal of General Virology vol 79 no 12 pp 3073ndash3077 1998

[4] M H M Noteborn G F de Boer D J van Roozelaar etal ldquoCharacterization of cloned chicken anemia virus DNAthat contains all elements for the infectious replication cyclerdquoJournal of Virology vol 65 no 6 pp 3131ndash3139 1991

[5] M F Ducatez A A Owoade J O Abiola and C P MullerldquoMolecular epidemiology of chicken anemia virus in NigeriardquoArchives of Virology vol 151 no 1 pp 97ndash111 2006

[6] M I Craig A Rimondi M Delamer et al ldquoMolecular char-acterization of chicken infectious anemia virus circulating inArgentina during 2007rdquo Avian Diseases vol 53 no 3 pp 331ndash335 2009

[7] K A Schat ldquoChicken infectious anemiardquo inDiseases of PoultryY M Saif H J Barnes J R Glisson A M Fadly L RMcDougald and E Swayne Eds pp 182ndash202 Iowa StateUniversity Press Ames Iowa USA 11th edition 2003

[8] G H Lai M K Lin Y Y Lien et al ldquoExpression andcharacterization of highly antigenic domains of chicken anemiavirus viral VP2 and VP3 subunit proteins in a recombinant Ecoli for sero-diagnostic applicationsrdquo BMC veterinary researchvol 9 p 161 2013

[9] H A Hussein M Z Sabry E A El-Ibiary M El-Safty and AI Abd El-Hady ldquoChicken infectious anaemia virus in Egyptmolecular diagnosis by PCR and isolation of the virus frominfected flocksrdquo Arab Journal of Biotechnology vol 5 no 2 pp263ndash274 2002

[10] M Hegazy F M Abdallah L K Abd-El Samie and A ANazim ldquoChicken infectious anemia virus (CIAV) in broilers andlaying hens in Sharkia Province Egyptrdquo Journal of AmericanScience vol 6 pp 752ndash758 2010

[11] MAMohamed ldquoChicken infectious anemia status in commer-cial broiler chickens flocks in assiut-upper Egypt occurrencemolecular analysis using PCR-RFLP and apoptosis effect onaffected tissuesrdquo International Journal of Poultry Science vol 9no 6 pp 591ndash598 2010

[12] D Todd K A Mawhinney and M S McNulty ldquoDetectionand differentiation of chicken anemia virus isolates by usingthe polymerase chain reactionrdquo Journal of ClinicalMicrobiologyvol 30 no 7 pp 1661ndash1666 1992

[13] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis forWindows 9598NTrdquoNucleic AcidsSymposium Series vol 41 pp 95ndash98 1999

[14] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[15] W Zhou B Shen B Yang et al ldquoIsolation and identification ofchicken infectious anemia virus in Chinardquo Avian Diseases vol41 no 2 pp 361ndash364 1997

[16] M R Islam R Johne R Raue D Todd and H MullerldquoSequence analysis of the full-length cloned DNA of a chickenanaemia virus (CAV) strain from Bangladesh evidence forgenetic grouping of CAV strains based on the deduced VP1amino acid sequencesrdquo Journal of Veterinary Medicine B vol49 no 7 pp 332ndash337 2002

[17] C J Snoeck G F Komoyo B P Mbee et al ldquoEpidemiologyof chicken anemia virus in Central African Republic andCameroonrdquo Virology Journal vol 9 pp 189ndash197 2012

[18] H E M Smuts ldquoNovel gyroviruses including chicken anaemiavirus in clinical and chicken samples from South AfricardquoAdvances in Virology vol 2014 Article ID 321284 7 pages 2014

[19] Z Hailemariam A R Omar M Hair-Bejo and T C GiapldquoDetection and characterization of chicken anemia virus fromcommercial broiler breeder chickensrdquo Virology Journal vol 5article 128 2008

[20] R W Renshaw C Soine T Weinkle et al ldquoA hypervariableregion in VP1 of chicken infectious anemia virus mediates rateof spread and cell tropism in tissue culturerdquo Journal of Virologyvol 70 no 12 pp 8872ndash8878 1996

[21] V L van Santen L Li F J Hoerr and L H Lauerman ldquoGeneticcharacterization of chicken anemia virus from commercialbroiler chickens in Alabamardquo Avian Diseases vol 45 no 2 pp373ndash388 2001

[22] D Todd A N J Scott N W Ball B J Borghmans andB M Adair ldquoMolecular basis of the attenuation exhibitedby molecularly cloned highly passaged chicken anemia virusisolatesrdquo Journal of Virology vol 76 no 16 pp 8472ndash8474 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

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Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

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Evolutionary BiologyInternational Journal of



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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

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Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Samples collected for the current study

Province Numbers of the flocks Type of samples Age of the flockSharkia Three Tissue homogenates 12ndash35 daysKafr El Sheikh One Tissue homogenate 25 daysMinoufiya One Liver 30 days

2 Materials and Methods

21 Samples Collection Tissue homogenates were collectedfrom Sharkia andKafr El Sheikh provincesThe homogenatescontained thymus loops bone marrow bursa of Fabriciusliver spleen and intestines samples Tissue homogenatesof Sharkia province were collected from three commercialbroiler flocks The flocks ranged in age from 12 to 35 daysrepresenting different breeds and localities in the provinceTissue homogenate of Kafr El Sheikh Province was onlycollected from one commercial broiler flock aged 25 daysFive liver samples were also collected from a commercialbroiler chicken flock aged 30 days from Minoufiya provinceAll flocks of collected samples were showing clinical signsand lesions indicative of CAV infection [10] The placesnumbers of flocks and age of flocks of selected samples arelisted in Table 1 All samples were stored frozen at minus70∘C forsubsequent DNA extraction

22 Sample Preparation Tissues were homogenized in amortar with sterile sand and phosphate-buffered saline Celldebris and sand were eliminated by centrifugation and thesupernatants were collected and stored at minus70∘C

23 DNA Extraction and PCR Amplification DNA wasextracted from the supernatant of liver and tissuehomogenates by QIAamp DNA mini kit (Qiagen IncValencia CA) according to the manufacturer instructionsThe oligonucleotide primers 51015840-GAC TGT AAG ATG GCAAGA CGA GCT C-31015840 and 51015840-GGC TGA AGG ATC CCTCAT TC-31015840 were used to amplify a 675 bp DNA fragmentof Vp1 [12] from nucleotides 823 to 1498 numbering iscorresponding to the Del-Ros strain (GenBank AF313470)The PCR assay was performed in a final volume of 50 120583LReddy-Mix 18120583L PCR grade water 1 120583L of each primer and5 120583L template The amplification was performed under thefollowing conditions one cycle of initial denaturation stepat 95∘C for 15min followed by 30 cycles of 95∘C for 1min56∘C for 1min and 72∘C for 1min representing denaturationannealing and extension steps respectively and finally onecycle of final extension step at 72∘C for 5min The amplifiedproducts were analyzed using electrophoresis unit Theywere loaded to 1 agarose stained by ethidium bromidevisualized under 304 nm ultraviolet (UV TransilluminatorMajor Science) and photographed by a gel documentationsystem using Canon power-shot camera

24 Nucleotide Sequence Analysis Sequencing was per-formed in both directions with virus specific primersSequences were analyzed using BioEdit program [13] This

program was also used to read the sequencing electrophero-grams to exclude nucleotide ambiguity The phylogeneticanalysis was based on the deduced amino acids of 579nucleotides encompassing hypervariable region of vp1 Thedetected sequences were compared with others deposited inGenBank by multiple alignment with the Clustal W includedin Bioedit software Phylogenetic relationships were evalu-ated by the Neighbor Joining method present in the MEGAversion 6 software [14] with 1000 bootstrap replicationsSequence data were submitted to GenBank with accessionnumbers KJ955377 toKJ955380 for the Egy-1 CAV to the Egy-4 CAV respectively

25 Attempted Isolation of the Virus Isolation of CAV fromthe tissue homogenate of the PCR-positive samples wasattempted in embryonated SPF eggs through yolk sac inoc-ulation [7] Tissue samples were prepared according to [15]Briefly the homogenate was mixed with an equal volume ofchloroform for 15min in a shaker Three times of repeatedfreezing and thawing were applied and then the homogenatewas centrifuged for 20min at 3000 rpm The supernatantswere used for SPF egg inoculation Each homogenate wasinjected in 5 SPF eggs (100 120583Legg) After 14 days all embryostissues were homogenized DNA extraction and PCR ampli-fication were carried out from the supernatant of these tissuehomogenates in the same condition as described above Thewhole isolation attempt was repeated once

3 Results

31 Samples and Clinical Signs All affected birds were atage of 12 to 35 days All affected flocks showed signs ofanemia generalized weakness depression pale comb andwattles growth retardation and high mortality rates Thepostmortem examination revealed pale and enlarged liverand spleen mild to severe thymus atrophy and atrophiedbursa of Fabricius

32 PCR Amplification Analysis of PCR amplification ofthe extracted DNA from tissue samples by agarose gelelectrophoresis indicated DNA bands of corrected size asexpected with a length of 675 bp The authenticity of PCRamplification was confirmed by the nucleotide sequencingAll of the liver samples from the flock in Minoufiya provinceshowed negative results Also all SPF embryo homogenateswere PCR negative

33 Nucleotide Sequence Analysis The four nucleotidesequences of detected CAV displayed a limited diversityTotal nucleotide variation among the sequences ranged from











4 Discussion
































5 Conclusion






CIA-1USAL147671


130SloveniaDQ016138

704AustraliaU654141


CL37ChileJQ3082131


CAV-BIndiaAY583756

G6JapanAB1194481






TR20JapanAB0274701

Conn-BUSAU695481

82-2JapanD319651

CAV-AIndiaAY583755




Del-RosUSAAF313470

ChinaAF448446




98D02152USAAF311890


26P4USAD100681

A2JapanAB0312961

75

51

64

97

0005

I

III

II





Acknowledgments



References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











4 Discussion
































5 Conclusion






CIA-1USAL147671


130SloveniaDQ016138

704AustraliaU654141


CL37ChileJQ3082131


CAV-BIndiaAY583756

G6JapanAB1194481






TR20JapanAB0274701

Conn-BUSAU695481

82-2JapanD319651

CAV-AIndiaAY583755




Del-RosUSAAF313470

ChinaAF448446




98D02152USAAF311890


26P4USAD100681

A2JapanAB0312961

75

51

64

97

0005

I

III

II





Acknowledgments



References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























5 Conclusion






CIA-1USAL147671


130SloveniaDQ016138

704AustraliaU654141


CL37ChileJQ3082131


CAV-BIndiaAY583756

G6JapanAB1194481






TR20JapanAB0274701

Conn-BUSAU695481

82-2JapanD319651

CAV-AIndiaAY583755




Del-RosUSAAF313470

ChinaAF448446




98D02152USAAF311890


26P4USAD100681

A2JapanAB0312961

75

51

64

97

0005

I

III

II





Acknowledgments



References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





CIA-1USAL147671


130SloveniaDQ016138

704AustraliaU654141


CL37ChileJQ3082131


CAV-BIndiaAY583756

G6JapanAB1194481






TR20JapanAB0274701

Conn-BUSAU695481

82-2JapanD319651

CAV-AIndiaAY583755




Del-RosUSAAF313470

ChinaAF448446




98D02152USAAF311890


26P4USAD100681

A2JapanAB0312961

75

51

64

97

0005

I

III

II





Acknowledgments



References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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