research article comparison of the effect of two...

Research ArticleComparison of the Effect of Two Purification Methods onthe Immunogenicity of Recombinant Outer Membrane ProteinH of Pasteurella multocida Serovar A1

Arunee Thanasarasakulpong1 Pichayanut Poolperm2 Weerapongse Tangjitjaroen1

Thanya Varinrak1 Takuo Sawada2 Dirk Pfeiffer13 and Nattawooti Sthitmatee1

1Faculty of Veterinary Medicine Chiang Mai University Chiang Mai 50100 Thailand2Laboratory of Veterinary Microbiology Nippon Veterinary and Life Science University Tokyo 180-8602 Japan3Veterinary Epidemiology Economics and Public Health Group Royal Veterinary College London AL9 7TA UK

Correspondence should be addressed to Nattawooti Sthitmatee drneawgmailcom

Received 13 October 2015 Accepted 27 December 2015

Academic Editor Francesca Mancianti

Copyright copy 2016 AruneeThanasarasakulpong et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Recombinant outer membrane protein H (rOmpH) of Pasteurella multocida strain X-73 can be purified using affinitychromatography but this adversely affects its immunogenicity The current study presents the results from an intervention studycomparing the immunogenicity of rOmpH purified using electroelution with rOmpH purified using affinity chromatography andnative OmpH purified using electroelution and a nonimmunized control group Chickens immunized with rOmpH purified usingelectroelution produced the highest ELISA antibody levels against P multocida strains Chickens in each of the 5 treatment groupswere split into two subgroups for challengewith two different Pmultocida strainsThe average number of adhesions to CEF cells wasstatistically significantly lower in sera from chickens immunizedwith rOmpHor nativeOmpHpurified using electroelution than inthose of the three other treatment groupsThe survival amongst chickens immunized with rOmpH or native OmpH purified usingelectroelution indicated high levels of protection In contrast survival probability was zero or low in the groups immunized withrOmpH purified using affinity chromatography and in the nonimmunized group These findings show that the rOmpH purifiedusing electroelution retains its immunogenicity and stimulates high levels of protection in chickens against P multocida infection

1 Introduction

Recombinant proteins with 6timesHis-tagged protein are rou-tinely purified by a Ni-NTA affinity chromatography asrecommended by their manufacturers However Luo et al[1] and Rimler [2] suggested that this process resulted in achange in the structure of the recombinant outer membraneprotein H (rOmpH) of avian Pasteurella multocida strain X-73 affecting its immunogenicity The OmpH a porin proteinis stable in the homotrimer form at room temperature andwas fully dissociated into monomers which correlated withthe unfolded or denatured form of protein after purifica-tion of the protein using a denatured condition of affinitychromatography In contrast Sthitmatee et al [3] successfullyimproved the immunogenicity of rOmpH using a hybrid

condition of affinity chromatography to purify rOmpHBut this method is unstable is of low reproducibility andresults in a high loss of protein yield [3] This suggests thataffinity chromatography may be unsuitable for purificationof this recombinant protein The electroelution method iswidely used for analytic purposes [4ndash6]Themethod employspolyacrylamide gel electrophoresis (PAGE) which is easy toperform and has high resolution and good reproducibilityThis system also has advantages in terms of having high load-ing capacity of sample protein and allowing easy monitoringof the elution process Previous studies using electroelutionto purify target proteins showed that the method provides aneffective purificationmethod for protection of immunogenic-ity of the target proteins [7ndash10] Interestingly the methodhas been used for purification of a native form of OmpH

Hindawi Publishing CorporationVeterinary Medicine InternationalVolume 2016 Article ID 2579345 7 pageshttpdxdoiorg10115520162579345

2 Veterinary Medicine International

Table 1 Experimental design and results of P multocida challenge of immunized and nonimmunized chickens

Treatmentgroups Type of immunogen

P multocida strain challenge subgroupNumber of survivorstotal ( protection)X-73 P-1059

1 rOmpH purified using electroelution 910 (90)lowast 810 (80)lowast

2 rOmpH purified using a denatured condition of affinitychromatography 210 (20) 310 (30)

3 Native OmpH purified using electroelution 1010 (100)lowast 1010 (100)lowast

4 Incomplete Freundrsquos adjuvant 05 (0) 05 (0)5 No immunization 05 (0) 05 (0)lowastSignificantly difference (119901 lt 005)

while completely protecting its immunogenicity [8] Thissuggests that the electroelution method could also be appliedfor purification of rOmpH The present intervention studyaimed at comparing the performance of the electroelutionand affinity chromatography methods for purification of therOmpH in terms of their effect on the immunogenicity of therecombinant protein

2 Materials and Methods

21 Experimental Design An intervention study was usedwith five treatment groups there were four immunizedgroups and one nonimmunized group (Table 1) The treat-ments included group 1 immunized with rOmpH purifiedusing electroelution group 2 immunized with rOmpH puri-fied using a denatured condition of affinity chromatography[1] group 3 immunized with native OmpH purified usingelectroelution [8] group 4 immunized with incompleteFreundrsquos adjuvant and group 5 a nonimmunized grouprespectively Each of the five treatment groups was dividedinto two subgroups one challenged with P multocida serovarA1 and the other with serovar A3 There were thereforein total 10 treatment-challenge subgroups Hisex brownchickens at the age of 21 weeks sourced from RPM Farmamp Feed Co Ltd Chiang Mai Thailand were used inthis study The outcome variables which were comparedbetween the treatment groups were immunological andclinical parameters The former consisted of serum antibodyand cell adhesion levels in response to infection challengeboth measured after vaccination and the latter of survivalof chickens The chickens were randomly allocated to the10 treatment-challenge subgroups with 10 chickens in eachof the six subgroups immunized with rOmpH or OmpHpurified using electroelution and 5 chickens in each of thefour other subgroups The group size of 10 was sufficientto detect a reduction in mortality in a pairwise comparisonfrom 100 to 40 at 95 confidence level and with 80power A group size of 10 in each of the OmpH or rOmpHimmunized and 5 in each of the two comparison groupsallowed for detection of a reduction in mortality from 100 to30 at 95 confidence level and with 80 statistical powerChickens in groups 1ndash3 were intramuscularly immunized twotimes at a 2-week interval with a total volume of 1mL of100 120583g rOmpH or OmpH emulsified with an equal volumeof incomplete Freundrsquos adjuvant (Sigma-Aldrich) specifically

group 1 with rOmpH purified using electroelution group 2with a denatured condition of affinity chromatography andgroup 3 with native OmpH purified using electroelutionChickens in group 4 were intramuscularly immunized withincomplete Freundrsquos adjuvant in PBS buffer and group 5was not immunized (Table 1) Chickens in the 4 immunizedgroups were immunized twice once on day 0 and againon day 14 All groups were intramuscularly challenged witheither approximately 2 times 106 cfumL or 43 times 106 cfumL oflive P multocida strains X-73 or P-1059 [3] respectively at 2weeks after the second immunization

Blood samples from each chicken were collected from thewing veins on days 0 7 14 21 and 28 after immunizationSera were assayed using western blot indirect ELISA andthe adhesion inhibition assay All chickens were observedevery day for clinical signs and behavioral changes duringthe experiments Once chickens showed clinical signs of thedisease they were euthanised according to the protocol inthe AVMA guideline for the euthanasia of animals version2013 [11] The experimental use of animals in this study wasapproved by the animal welfare and laboratory animal ethicscommittee of the Faculty of Veterinary Medicine ChiangMai University Chiang Mai Thailand (approval numberR152555)

22 Bacterial Strains Gene and Plasmid The challengeexperiments were conducted using P multocida strainsX-73 (serovar A1 ATCC15742) and P-1059 (serovar A3ATCC11039) which as major etiologic strains of fowl choleraare widely considered to be appropriate for assessing theprotection of chickens against infection with all P multocidastrains They were grown in brain heart infusion broth (BHIMerck Darmstadt Germany) for 6 h at 37∘C Then thebacteria were subcultured onto a blood agar and incubatedat 37∘C for 18 h E coli strain PQE-ompH which carried the6timesHistidine tag fused ompHgene of thePmultocida strainX-73 plasmid in the E coli strain M15 from our previous study[3] was grown in Luria-Bertani (LB) broth or on LB agarcontaining 100 120583gmL ampicillin and 25120583gmL kanamycin(Sigma-Aldrich St Louis MO USA) at 37∘C

23 Purification of Native OmpH Native OmpH was pre-pared using the electroelutionmethod as described elsewhere[8] Briefly crude capsular extract (CCE) was prepared usingthe saline extraction method as described previously [8]

Veterinary Medicine International 3

Then target 39 kDa protein of native OmpH was purifiedby electroelution (electroelution electrophoresis apparatusATTO) in 20mM Tris base 150mM glycine and 001 SDSbuffer at 100V for 1 h in an iceboxThe conditions for proteincollection were 200min for delay time 2min for EP time100 s for filling time 120 s for collecting time and 15mAfor electrical current The eluted native OmpH was passedthrough the detergent removing minicolumn (Ampure DTAmersham Japan)Then the total protein in the supernatantwas quantified using the BCA protein assay kit (PierceRockford IL USA) and the eluted protein was kept at minus20∘Cuntil use

24 Preparation and Purification of rOmpH TherOmpHwasexpressed via E coli strain PQE-ompH as described in ourprevious study [3] Then the expression of the recombinantprotein was induced by the addition of isopropyl-120573-D-thiogalactopyranoside (IPTG Amresco Solon OH USA)to a final concentration of 1mM and continually incubatedunder the same conditions for a further 5 h Finally thebacterial cells were harvested by centrifugation at 4000timesg at4∘C for 20min The supernatant was discarded and the cellpellets were stored in minus20∘C for further utilization

E coli cell pellets were lysed and purified using theelectroelution method or a denatured condition of affinitychromatography [1] Purification of the recombinant proteinusing the electroelutionmethodwas explained as followsThe5 g wet weight of cell pellets was lysed in 10mL of a nativelysis buffer (50mM NaH

2PO4 300mM NaCl and 10mM

imidazole pH80)The solutionwas gentlymixed at 4∘C andthen the homogenate was centrifuged at 10000timesg at 4∘CThesupernatant containing 1500 120583g of total protein was run ona preparative 125 sodium dodecyl sulfate polyacrylamidegel column (10mm stacking gel and 30mm separating gel)in a sample buffer (4 SDS 50mM Tris 20 of glyceroland 0005 of bromophenol blue) using the electroelutionapparatus (Nativen ATTO Tokyo Japan)The conditions forprotein collection were 200min for delay time 2min for EPtime 100 s for filling time 120 s for collecting time and 15mAfor electrical current Protein fractions were collected inbuffer (371mMTris 5 sucrose pH 88) and the total proteinwas quantified using the BCAprotein assay kit (Pierce) beforebeing kept at minus20∘C

25 Protein Fraction Analyses Each 10 120583g protein fractionwas identified on the basis of presence of the target proteinby 125 SDS-PAGE according to the Laemmli method[12] and then subjected to western blotting Protein frac-tions were transferred onto nitrocellulose membrane andimmunostained with an anti-HisG-HRP antibody (Invitro-gen Carlsbad CA USA) After incubation with antibodiesthe membranes were washed thoroughly with PBST Theprotein bands were visualized following incubation with331015840-diaminobenzidine (DAB Invitrogen) as a chromogenicsubstrate

26 Determination of Antibody Responses Specific anti-body responses of the chicken sera were determinedthrough measuring the Immunoglobulin Y (IgY) titers

using a commercial indirect ELISA test kit for fowl cholera(ProFLOK Synbiotics Kansas City MO USA) The plateswere evaluated using an ELISA plate reader (Immuno MiniNJ 2300 Intermed Japan) and the average sample perpositive (SP) ratio of each group was calculated accordingto the manufacturerrsquos recommendation The SP ratios werecalculated according to the following equation SP ratio() = [corrected optical density of a samplecorrected opticaldensity of a positive reference serum]There is no quantitativetest sensitivity and specificity information available for thistest

27 Chicken Embryo Fibroblast (CEF) Cell Culture The CEFcells were obtained from 10-day-embryonated chicken eggs(The Upper Northern Veterinary Research and DevelopmentCenterHangChat LamPangThailand) Approximately 48 hbefore the experiment a total of 25 times 105 cellsmL in 2mLDulbeccorsquos Modified Eagle Medium (DMEM Invitrogen)supplemented with 5 fetal bovine serum (Invitrogen) 1L-glutamine (Invitrogen) and 100UmL of penicillin andstreptomycin (Invitrogen) were seeded into 35mm Corningculture dishes containing 22 times 22mm cover slips in thebottom of the well The dishes were incubated at 37∘C with5 CO

2 After the incubation the dishes were washed three

times with 2mL of sterile PBS pH 74 and used for theadhesion inhibition assay

28 Adhesion Inhibition Assay The adhesion inhibition assaywas modified based on a previous study [8] Briefly Pmultocida strains were grown separately on blood agar at37∘C for 18 h and were resuspended in sterile PBS andsuspension turbidity was adjusted to 05 of McFarland Stan-dard (approximately 28 times 108 cfumL) at the wavelength of600 nm The day after vaccination with the highest averageSP value in the indirect ELISA across all serum sampleswas identified and samples from that day were pooledwithin each of the 10 treatment-challenge groups and usedfor this assay To represent the bacterial challenge in vitrofor each of the 10 treatment-challenge groups 2mL of thebacterial suspension was added to 3mL of pooled chickenserum and incubated at 37∘C for 1 h After the incubationthe resulting suspension was inoculated onto the monolayerof CEF cells and incubated at 37∘C with 5 CO

2for 1 h

Nonadherent bacteria were removed by washing with 2mL ofsterile PBS The washing step was repeated for 4 times Afterwashing the cover slips were fixed with 4 formaldehydeand stained with Wright Giemsa solution (Sigma-Aldrich)The cover slips were examined under a light microscope with1000x power of magnification For each of the treatment-challenge subgroups 100 CEF cells with intact structure wereselected and the number of adhering bacteria was countedThe selection of CEF cells occurred randomly by scanning amagnification field from the left to the right and from the topto the bottom of the cover slips The counts for each selectedCEF cell were performed twice and the mean was used in theanalysis

29 Experimental Infection in Chickens After allowing twoweeks following the second immunization for chickens to


M 1 2 3 4

(a)

M 1 2 3 4

(b)

Figure 1 The fractions of rOmpH purified by electroelution were analyzed using SDS-PAGE (a) and probed with anti-HisG-HRP antibody(b) Lane M cell lysates of the E coli host Lanes 1ndash4 rOmpH fractions purified using electroelution

develop an immune response chickens in all groups werechallenged with the bacteria by intramuscular injection of1mL of bacterial suspension containing 2 times 106 cfumL ofstrain X-73 or 43 times 106 cfumL of P-1059 [3] respectivelyThe birds were examined for clinical signs over a 7-daypostchallenge period and mortality was recorded

210 Statistical Analysis The results were analyzed usingStata SE 131 software (StataCorp LP College station TXUSA) and IBM SPSS Statistics version 22 No statisticalanalysis was performed for the antibody response data as onlysummary statistics were available A general linear modelanalysis was used to compare the average adhesion inhibitionof bacteria to CEF cells counts between treatment groups andin vitro infection challenge groups as well as their interactionThe means between different groups were compared usingsimple contrasts The survival of chickens was comparedbetween different treatment and in vivo infection challengegroups using binary logistic regression Exact estimation wasused given the small sample sizes in this study

3 Results

31 Expression and Purification of rOmpH E coli cell wholecell lysates showed an overexpressed band at approximately39 kDa on sodium dodecyl sulfate polyacrylamide gel elec-trophoresis (SDS-PAGE Figure 1) The rOmpH fractionsfrom the electroelution apparatus which was employedin order to check whether the eluted fractions had beenextracted showed a single protein band with the same targetmolecular mass as shown in Figure 1 The western blottingprobed with anti-6timesHistidine tagged-antibody also con-firmed the overexpressed band of rOmpH which was taggedwith the 6timesHistidine as shown in Figure 1 Additionally all

75kDa63kDa48kDa

35kDa25kDa

OmpH (39kDa)

M 1 2 3 4 5

Figure 2 SDS-PAGE of proteins used in this study Lanes Mmolecular mass standards 1 whole cell lysate of strain X-732 prepurified rOmpH 3 rOmpH purified by electroelution 4rOmpH purified by a hybrid condition of affinity chromatographyand 5 native OmpH of strain X-73 purified by electroelution

the proteins used in this study were analyzed using SDS-PAGE (Figure 2) The molecular mass of those 3 proteinsnative OmpH and the two types of rOmpH produced usingdifferent purification methods were identical with approxi-mately 39 kDa

32 Antibody Responses after Vaccination It is to be notedthat the findings in relation to antibody data are based onvisual analysis of the data since only summary statisticswere available The levels of serum antibody in the chickensimmunized with native OmpH or rOmpH both purifiedusing the electroelution method increased following the firstand second immunizations (Figure 3) Immune responses tonative OmpH reached a maximal point 7 days after first doseand began to plateau after 14 days but responses were also


Native OmpHrOmpH-affinity chromatographyrOmpH-electroelutionFreundrsquos incomplete adjuvantNo immunization

0 7 14 21 28Days after immunization

020

040

060

080

100

120

SP

valu

e

Figure 3 The temporal pattern of the average SP value of chickensera (including standard error) based on indirect ELISA (ProFLOK)for each of the five treatment groups (first vaccination on day 0 andthe second on day 14)

slightly increased at days 21 and 28 In contrast rOmpHreached a maximal point after 7 days and began to plateauat day 7 after first dose Moreover responses were alsoslightly increased at day 21 (rOmpH) but do not appearto be significantly different to the responses at day 14 andtherefore were considered to have reached a maximum atday 14 In contrast low antibody levels were observed inthe chickens immunized with the rOmpH purified usingaffinity chromatography those receiving incomplete Freundrsquosadjuvant and the nonimmunized groups

33 Bacterial Adhesion following In Vitro Challenge with Pmultocida Strains The general linear model analysis indi-cates that the mean number of adherent bacteria differsamongst treatment groups (119901 lt 0001) and between thetwo challenge strains (119901 lt 0001) There was no statisticallysignificant interaction between treatment group and chal-lenge strain (119901 = 007) Compared with the counts for thenonvaccinated group the samples from chickens immunizedwith Freundrsquos incomplete adjuvant had average bacterialadhesion counts per CEF which were lower by 89 (95CI 59ndash119) those immunized with rOmpH purified usingaffinity chromatography were lower by 150 (95 CI 12ndash18)those immunized with rOmpH purified using electroelutionwere lower by 35 (95 CI 324ndash385) and those immunizedwith OmpH purified using electroelution were lower by 45(95 CI 426ndash482) The pooled sera challenged in vitro withP multocida strain P-1059 had on average 41 (95CI 23ndash59)more adherent bacteria per CEF cell than those challengedwith X-73The distributions of counts of bacteria adherent toCEF cells for each of the 10 treatment-challenge groups areshown in Figure 4

lowast

lowast

Adhe

sion

coun

ts

No

imm

uniz

atio

n

Freu

ndrsquos

inco

mpl

ete a

djuv

ant

rOm

pH

(affi

nity

chro

mat

ogra

phy)

rOm

pH

(ele

ctro

elutio

n)

Nat

ive O

mpH

(e

lect

roelu

tion)

Treatment groupStrainP-1059X-73

0

20

40

60

80

100

120

Figure 4 Box-and-whisker plot of counts of adhesions to CEF cellsby treatment group and P multocida challenge strains

34 Survival following In Vivo Infection Challenge with P mul-tocida Strains Amongst chickens developing clinical diseaseclinical signs were observed from 12 hours following bacterialchallenge There was no statistically significant differencein survival between the two challenge strain groups andthis effect did not vary between treatment groups (medianunbiased estimate of odds ratio = 1 exact 95 CI 019ndash51) There was a statistically significant difference in survivalbetween treatment groups Using nonvaccinated chickens asthe reference group there was a higher survival proportionin chickens immunized with either OmpH (median unbiasedestimate of odds ratio = 238 exact 95 CI 33ndashinfinity) orrOmpH purified using electroelution against challenge withone of the two Pmultocida strains (median unbiased estimateof odds ratio = 55 exact 95 CI 9ndashinfinity) The other twogroups did not differ in their survival from the referencegroup Table 1 shows the proportions of surviving chickensfor each treatment and P multocida strain challenge group

4 Discussion

The structural integrity of the expressed protein is one ofthe main concerns in recombinant protein production Thisis due partly to the original protein structure having animportant role in inducing specific antibodies The antigenicepitope and structure of the recombinant protein must alsobe conserved during the purification process Electroelution


is used to extract a particular protein of interest from anelectrophoresis gel by applying an electric current [6 13]The method is considered to be an effective purificationmethod for separating a small target band of proteins froma crude whole protein sample [7ndash10] Other chromatographymethods are less able to uniquely separate the target proteinband [6] The electroelution method uses polyacrylamidegel electrophoresis (PAGE) which allows effective separationof the target protein band from a crude original protein[6 13] Indeed PAGE is routinely used to determine proteinor nucleotide purity Amongst its advantages is also theabsence of sodium dodecyl sulfate (SDS) and urea in the gelcomposition In the current study electroelution was appliedto purify the rOmpH from cell lysate in order to protect theimmunogenicity of the protein

Outer membrane protein H (OmpH) is a porin proteinwhich is considered to be highly conserved among Gram-negative bacteria including P multocida strains [14] A nativeform of OmpH is a homotrimer of approximately 110 kDawhile a monomeric form of this protein can be obtain byinduced denaturation and ranges from 34 to 42 kDa Thevariation in the size of the monomer depends on the serotypeand the electrophoretic system used for the analysis [1 15 16]Chevalier et al [15] demonstrated the use of size exclusionchromatography to purify the native form of OmpH and suc-cessfully protected the immunogenicity of the protein Sub-sequently Luo et al [1] employed the same method and usedthe protein as an immunogen in chickens The native formof OmpH produced effective protection in chickens againsthomologous challenge-exposure Recombinant OmpH of Pmultocida strain X-73 has been cloned and expressed by Luoet al [1] The rOmpH was purified by a denatured conditionof affinity chromatography and characterized the immuno-genicity The protection in chickens conferred by immuniza-tion with rOmpH in that study was low when comparedwith a native form of OmpH Luo et al [1] suggested thatthe structure of the recombinant outer membrane proteinH (rOmpH) of avian P multocida strain X-73 had changedand affected its immunogenicity following purification by adenatured condition of affinity chromatography Accordingto previous reports on immunization with bacterial porins inanimal models the trimeric or native conformation of porinis considered essential for induction of protective immunity[1 2] Until now there has been only one application of theelectroelution method for purification of a native form ofOmpH (Borrathybay et al 2003) however the applicationfor purification of rOmpH has not been demonstrated yetBorrathybay et al [8] attempted to purify a native form ofOmpH of P multocida strain P-1059 from crude capsularextract The purified OmpH provided effective protection inchicken against challenge with different P multocida strainsThis suggested that the electroelution method used in thatstudy had no adverse effect on the immunogenicity of OmpHafter purification According to the results from the currentstudy the protection conferred by immunizationwithOmpHor rOmpH purified using the electroelution method resultedin a higher level of protection amongst chickens comparedwith other purification methods This indicates that theelectroelutionmethod can be used for purification of rOmpH

without adversely affecting its immunogenicity Furthermoregiven that there was no statistically significant differencein the performance of native OmpH and rOmpH bothpurified using electroelution it can be concluded that therecombinant OmpH of P multocida can be used as effectivelyas the native one

The ability to induce cross-immunity among P multocidaserovars is important in the development of poultry vaccinesSimilar to this study Sthitmatee et al [3] and Borrathybayet al [8] also demonstrated that OmpH and rOmpH of Pmultocida strains are cross-protective immunogens againstavianPmultocida strainsThemethodused for purification ofthe protein influences its immunogenicity including its cross-protection potential This is important since a natural fowlcholera outbreak can be caused by strains that are differentfrom the vaccination strain or may involve multiple strainsA previous study suggested that the natural expression of theantigen responsible for cross-protection is limited under thein vitro growth conditions during the proliferation process[17]

The current study demonstrates that both native OmpHand rOmpH purified using electroelution induce effectivein vitro antibody protection that inhibits the adhesion oftwo common avian P multocida strains to CEF cells Incontrast the antibodies induced by rOmpH purified bya denatured condition of affinity chromatography resultedin a high number of adhesions of bacteria to CEF cellsThis result provides strong evidence that the electroelutionwas successful at refolding the protein conformation Inaccordance with the previous study [8] the native formof OmpH induced an efficient antibody which inhibitedthe bacterium from adhering to CEF cells Moreover lowamounts of OmpH in bacterial capsule affected the cross-protectivity [18] However the method requires further basicbiochemistry coupled with bioinformatics tools to verify theprotein structure The result was confirmed in the in vivochallenge experiment where survival in the groups immu-nized with rOmpH purified using electroelution was 85compared with 25 in the groups immunized with rOmpHpurified using affinity chromatography These in vitro andin vivo results demonstrate the potential of rOmpH purifiedusing electroelution for protection of chickens against Pmultocida infection

5 Conclusion

The rOmpH purified using the electroelution method retainsits immunogenicity as demonstrated by being able to inducespecific antibodies against avian P multocida strains Itsuccessfully protected against homologous strain challenge asmeasured by in vitro and in vivo challenge with two differentavian P multocida strains The rOmpH purified using theaffinity chromatographymethod achieved poor protection inthe challenge experiment

Conflict of Interests

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


Acknowledgment

This research (Grant no P-10-11170) was financially sup-ported by theNational Science and TechnologyDevelopmentAgency (NSTDA) Ministry of Science and TechnologyThailand

References

[1] Y Luo J R Glisson M W Jackwood et al ldquoCloning andcharacterization of the major outer membrane protein gene(ompH) of Pasteurella multocida X-73rdquo Journal of Bacteriologyvol 179 no 24 pp 7856ndash7864 1997

[2] R B Rimler ldquoPurification of a cross-protective antigen fromPasteurella multocida grown in vitro and in vivordquo Avian Dis-eases vol 45 no 3 pp 572ndash580 2001

[3] N Sthitmatee S Numee E Kawamoto et al ldquoProtection ofchickens from fowl cholera by vaccination with recombinantadhesive protein of Pasteurella multocidardquo Vaccine vol 26 no19 pp 2398ndash2407 2008

[4] B Chellan P S Appukuttan andNN Jayakumari ldquoElectroelu-tion of lipoprotein(a) [Lp(a)] from native polyacrylamide gelsa new simple method to purify Lp(a)rdquo Journal of Biochemicaland Biophysical Methods vol 68 no 1 pp 43ndash53 2008

[5] P Sa-Pereira J Duarte and M Costa-Ferreira ldquoElectroelutionas a simple and fast protein purification method isolation of anextracellular xylanase fromBacillus sp CCMI 966rdquoEnzyme andMicrobial Technology vol 27 no 1-2 pp 95ndash99 2000

[6] M Shoji M Kato and H Shuichi ldquoElectrophoretic recovery ofproteins from polyacrylamide gelrdquo Journal of ChromatographyA vol 698 no 1-2 pp 145ndash162 1995

[7] P J Barrell O W Liew and A J Conner ldquoExpressing anantibacterial protein in bacteria for raising antibodiesrdquo ProteinExpression and Purification vol 33 no 1 pp 153ndash159 2004

[8] E Borrathybay T Sawada Y Kataoka et al ldquoA 39 kDa proteinmediates adhesion of avian Pasteurella multocida to chickenembryo fibroblast cellsrdquo Veterinary Microbiology vol 97 no 3-4 pp 229ndash243 2003

[9] U Kaur S Khurana U N Saikia and M L Dubey ldquoImmuno-genicity and protective efficacy of heparan sulphate bindingproteins of Entamoeba histolytica in a guinea pig model ofintestinal amoebiasisrdquo Experimental Parasitology vol 135 no3 pp 486ndash496 2013

[10] A R Pinto C G P Beyrodt R A M Lopes and C L BarbierildquoIdentification of a 30 kDa antigen from Leishmania (L) chagasiamastigotes implicated in protective cellular reponses in amurine modelrdquo International Journal for Parasitology vol 30no 5 pp 599ndash607 2000

[11] American Veterinary Medical Association AVMA Guidelinesfor the Euthanasia of Animals American Veterinary MedicalAssociation Press Schaumburg Ill USA 2013

[12] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[13] M G Harrington ldquoPurification procedures electrophoreticmethods elution of protein from gelsrdquo in Guide to ProteinPurification Methods in Enzymology M P Deutscher Ed pp488ndash498 Academic Press San Diego Calif USA 1990

[14] T Hatfaludi K Al-Hasani J D Boyce and B Adler ldquoOutermembrane proteins of Pasteurella multocidardquoVeterinaryMicro-biology vol 144 no 1-2 pp 1ndash17 2010

[15] G Chevalier H Duclohier D Thomas E Shechter and HWroblewski ldquoPurification and characterization of protein Hthe major porin of Pasteurella multocidardquo Journal of Bacteriol-ogy vol 175 no 1 pp 266ndash276 1993

[16] A Lubke L Hartmann W Schroder and E Hellmann ldquoIso-lation and partial characterization of the major protein of theouter membrane of Pasteurella haemolytica and Pasteurellamultocidardquo Zentralblatt fur Bakteriologie vol 281 no 1 pp 45ndash54 1994

[17] P A Rebers and K L Heddleston ldquoFowl cholera induction ofcross-protection in turkeys with bacterins prepared from host-passaged Pasteurella multocidardquo Avian Diseases vol 21 no 1pp 50ndash56 1977

[18] N Sthitmatee T Yano K N Lampang C Suphavilai YKataoka and T Sawada ldquoA 39-kDa capsular protein is amajor cross-protection factor as demonstrated by protection ofchickens with a live attenuated Pasteurella multocida strain ofP-1059rdquo Journal of Veterinary Medical Science vol 75 no 7 pp923ndash928 2013

Submit your manuscripts athttpwwwhindawicom

Veterinary MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International



International Journal of

Microbiology


AnimalsJournal of

EcologyInternational Journal of


PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of


Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

Biotechnology Research International


Agronomy




Journal of Parasitology Research

Hindawi Publishing Corporation httpwwwhindawicom


Volume 2014

Zoology

GenomicsInternational Journal of


InsectsJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


VirusesJournal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of



Case Reports in Veterinary Medicine


Table 1 Experimental design and results of P multocida challenge of immunized and nonimmunized chickens

Treatmentgroups Type of immunogen

P multocida strain challenge subgroupNumber of survivorstotal ( protection)X-73 P-1059

1 rOmpH purified using electroelution 910 (90)lowast 810 (80)lowast

2 rOmpH purified using a denatured condition of affinitychromatography 210 (20) 310 (30)

3 Native OmpH purified using electroelution 1010 (100)lowast 1010 (100)lowast

4 Incomplete Freundrsquos adjuvant 05 (0) 05 (0)5 No immunization 05 (0) 05 (0)lowastSignificantly difference (119901 lt 005)

while completely protecting its immunogenicity [8] Thissuggests that the electroelution method could also be appliedfor purification of rOmpH The present intervention studyaimed at comparing the performance of the electroelutionand affinity chromatography methods for purification of therOmpH in terms of their effect on the immunogenicity of therecombinant protein

2 Materials and Methods

21 Experimental Design An intervention study was usedwith five treatment groups there were four immunizedgroups and one nonimmunized group (Table 1) The treat-ments included group 1 immunized with rOmpH purifiedusing electroelution group 2 immunized with rOmpH puri-fied using a denatured condition of affinity chromatography[1] group 3 immunized with native OmpH purified usingelectroelution [8] group 4 immunized with incompleteFreundrsquos adjuvant and group 5 a nonimmunized grouprespectively Each of the five treatment groups was dividedinto two subgroups one challenged with P multocida serovarA1 and the other with serovar A3 There were thereforein total 10 treatment-challenge subgroups Hisex brownchickens at the age of 21 weeks sourced from RPM Farmamp Feed Co Ltd Chiang Mai Thailand were used inthis study The outcome variables which were comparedbetween the treatment groups were immunological andclinical parameters The former consisted of serum antibodyand cell adhesion levels in response to infection challengeboth measured after vaccination and the latter of survivalof chickens The chickens were randomly allocated to the10 treatment-challenge subgroups with 10 chickens in eachof the six subgroups immunized with rOmpH or OmpHpurified using electroelution and 5 chickens in each of thefour other subgroups The group size of 10 was sufficientto detect a reduction in mortality in a pairwise comparisonfrom 100 to 40 at 95 confidence level and with 80power A group size of 10 in each of the OmpH or rOmpHimmunized and 5 in each of the two comparison groupsallowed for detection of a reduction in mortality from 100 to30 at 95 confidence level and with 80 statistical powerChickens in groups 1ndash3 were intramuscularly immunized twotimes at a 2-week interval with a total volume of 1mL of100 120583g rOmpH or OmpH emulsified with an equal volumeof incomplete Freundrsquos adjuvant (Sigma-Aldrich) specifically

group 1 with rOmpH purified using electroelution group 2with a denatured condition of affinity chromatography andgroup 3 with native OmpH purified using electroelutionChickens in group 4 were intramuscularly immunized withincomplete Freundrsquos adjuvant in PBS buffer and group 5was not immunized (Table 1) Chickens in the 4 immunizedgroups were immunized twice once on day 0 and againon day 14 All groups were intramuscularly challenged witheither approximately 2 times 106 cfumL or 43 times 106 cfumL oflive P multocida strains X-73 or P-1059 [3] respectively at 2weeks after the second immunization

Blood samples from each chicken were collected from thewing veins on days 0 7 14 21 and 28 after immunizationSera were assayed using western blot indirect ELISA andthe adhesion inhibition assay All chickens were observedevery day for clinical signs and behavioral changes duringthe experiments Once chickens showed clinical signs of thedisease they were euthanised according to the protocol inthe AVMA guideline for the euthanasia of animals version2013 [11] The experimental use of animals in this study wasapproved by the animal welfare and laboratory animal ethicscommittee of the Faculty of Veterinary Medicine ChiangMai University Chiang Mai Thailand (approval numberR152555)

22 Bacterial Strains Gene and Plasmid The challengeexperiments were conducted using P multocida strainsX-73 (serovar A1 ATCC15742) and P-1059 (serovar A3ATCC11039) which as major etiologic strains of fowl choleraare widely considered to be appropriate for assessing theprotection of chickens against infection with all P multocidastrains They were grown in brain heart infusion broth (BHIMerck Darmstadt Germany) for 6 h at 37∘C Then thebacteria were subcultured onto a blood agar and incubatedat 37∘C for 18 h E coli strain PQE-ompH which carried the6timesHistidine tag fused ompHgene of thePmultocida strainX-73 plasmid in the E coli strain M15 from our previous study[3] was grown in Luria-Bertani (LB) broth or on LB agarcontaining 100 120583gmL ampicillin and 25120583gmL kanamycin(Sigma-Aldrich St Louis MO USA) at 37∘C

23 Purification of Native OmpH Native OmpH was pre-pared using the electroelutionmethod as described elsewhere[8] Briefly crude capsular extract (CCE) was prepared usingthe saline extraction method as described previously [8]














2for 1 h




M 1 2 3 4

(a)

M 1 2 3 4

(b)




3 Results


75kDa63kDa48kDa

35kDa25kDa

OmpH (39kDa)

M 1 2 3 4 5







020

040

060

080

100

120

SP

valu

e




lowast

lowast

Adhe

sion

coun

ts

No

imm

uniz

atio

n

Freu

ndrsquos

inco

mpl

ete a

djuv

ant

rOm

pH

(affi

nity

chro

mat

ogra

phy)

rOm

pH

(ele

ctro

elutio

n)

Nat

ive O

mpH

(e

lect

roelu

tion)


0

20

40

60

80

100

120



4 Discussion








5 Conclusion





Acknowledgment


References


























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of



















2for 1 h




M 1 2 3 4

(a)

M 1 2 3 4

(b)




3 Results


75kDa63kDa48kDa

35kDa25kDa

OmpH (39kDa)

M 1 2 3 4 5







020

040

060

080

100

120

SP

valu

e




lowast

lowast

Adhe

sion

coun

ts

No

imm

uniz

atio

n

Freu

ndrsquos

inco

mpl

ete a

djuv

ant

rOm

pH

(affi

nity

chro

mat

ogra

phy)

rOm

pH

(ele

ctro

elutio

n)

Nat

ive O

mpH

(e

lect

roelu

tion)


0

20

40

60

80

100

120



4 Discussion








5 Conclusion





Acknowledgment


References


























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







M 1 2 3 4

(a)

M 1 2 3 4

(b)




3 Results


75kDa63kDa48kDa

35kDa25kDa

OmpH (39kDa)

M 1 2 3 4 5







020

040

060

080

100

120

SP

valu

e




lowast

lowast

Adhe

sion

coun

ts

No

imm

uniz

atio

n

Freu

ndrsquos

inco

mpl

ete a

djuv

ant

rOm

pH

(affi

nity

chro

mat

ogra

phy)

rOm

pH

(ele

ctro

elutio

n)

Nat

ive O

mpH

(e

lect

roelu

tion)


0

20

40

60

80

100

120



4 Discussion








5 Conclusion





Acknowledgment


References


























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of









020

040

060

080

100

120

SP

valu

e




lowast

lowast

Adhe

sion

coun

ts

No

imm

uniz

atio

n

Freu

ndrsquos

inco

mpl

ete a

djuv

ant

rOm

pH

(affi

nity

chro

mat

ogra

phy)

rOm

pH

(ele

ctro

elutio

n)

Nat

ive O

mpH

(e

lect

roelu

tion)


0

20

40

60

80

100

120



4 Discussion








5 Conclusion





Acknowledgment


References


























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of












5 Conclusion





Acknowledgment


References


























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







Acknowledgment


References


























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of













Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of






research article comparison of the effect of two...

Documents