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Contents lists available at ScienceDirect

Vaccine

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scaridia galli infection influences the development of both humoralnd cell-mediated immunity after Newcastle Disease vaccination inhickens

anne Pleidrupa,1, Tina S. Dalgaarda,1, Liselotte R. Norupa, Anders Perminb,orben W. Schoub, Kerstin Skovgaardc, Dorte F. Vadekærc, Gregers Jungersenc,oul Sørensend, Helle R. Juul-Madsena,∗

Department of Animal Science, Aarhus University, DK-8830 Tjele, DenmarkDHI, Environment and Toxicology, Agern Allé 5, DK-2970 Hørsholm, DenmarkDTU Veterinary, National Veterinary Institute, Division of Veterinary Diagnostics and Research, Technical University of Denmark, Bülowsvej 27, DK-1870rederiksberg C, DenmarkDepartment of Molecular Biology and Genetics, Aarhus University, DK-8830 Tjele, Denmark

r t i c l e i n f o

rticle history:eceived 18 July 2013eceived in revised form7 September 2013ccepted 6 November 2013vailable online xxx

eywords:hicken. gallih2 cytokinesejunumewcastle disease vaccinerotective immunity.

a b s t r a c t

Potent vaccine efficiency is crucial for disease control in both human and livestock vaccination pro-grammes. Free range chickens and chickens with access to outdoor areas have a high risk of infectionwith parasites including Ascaridia galli, a gastrointestinal nematode with a potential influence on theimmunological response to vaccination against other infectious diseases. The purpose of this study wasto investigate whether A. galli infection influences vaccine-induced immunity to Newcastle Disease (ND)in chickens from an MHC-characterized inbred line. Chickens were experimentally infected with A. galliat 4 weeks of age or left as non-parasitized controls. At 10 and 13 weeks of age half of the chickens wereND-vaccinated and at 16 weeks of age, all chickens were challenged with a lentogenic strain of Newcastledisease virus (NDV). A. galli infection influenced both humoral and cell-mediated immune responses afterND vaccination. Thus, significantly lower NDV serum titres were found in the A. galli-infected group ascompared to the non-parasitized group early after vaccination. In addition, the A. galli-infected chickensshowed significantly lower frequencies of NDV-specific T cells in peripheral blood three weeks after thefirst ND vaccination as compared to non-parasitized chickens. Finally, A. galli significantly increased localmRNA expression of IL-4 and IL-13 and significantly decreased TGF-�4 expression in the jejunum two
weeks after infection with A. galli. At the time of vaccination (six and nine weeks after A. galli infection) thelocal expression in the jejunum of both IFN-� and IL-10 was significantly decreased in A. galli-infectedchickens. Upon challenge with the NDV LaSota strain, viral genomes persisted in the oral cavity fora slightly longer period of time in A. galli-infected vaccinees as compared to non-parasitized vaccinees.However, more work is needed in order to determine if vaccine-induced protective immunity is impaired
ns.
in A. galli-infected chicke
. Introduction

Worldwide, vaccination is a cost-effective intervention usedor control of a wide range of infectious diseases. However, there

Please cite this article in press as: Pleidrup J, et al. Ascaridia galli infectiimmunity after Newcastle Disease vaccination in chickens. Vaccine (2013)

s increasing evidence that parasite infections confound immuneesponses to third party antigens and may interfere with vaccinefficacy. Thus, in helminth-infected humans, a reduced humoralnd cellular immune response was observed after cholera [1],

∗ Corresponding author. Tel.: +45 87157837.E-mail address: [email protected] (H.R. Juul-Madsen).

1 Contributed equally.

264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.vaccine.2013.11.034

© 2013 Elsevier Ltd. All rights reserved.

bacillus Calmette-Guérin [2,3] and tetanus toxoid vaccination [4,5].In some cases, drug-induced clearance of the helminth infectionsignificantly improved vaccine-specific responses [1,2]. A negativeimpact of a persistent helminth infection on vaccine efficacy wasfurthermore described in a porcine model using co-infection withMycoplasma hyopneumoniae and Ascaris suum [6]. Pigs were contin-uously reinfected with A. suum eggs and subsequently immunizedwith killed M. hyopneumoniae. Both after immunizations and afterchallenge with live M. hyopneumoniae, the A. suum-infected ani-mals developed a significantly lower antibody response against M.hyopneumoniae than the non-parasitized animals.

on influences the development of both humoral and cell-mediated, http://dx.doi.org/10.1016/j.vaccine.2013.11.034

A common parasite species in poultry is Ascaridia galli, a gas-trointestinal nematode infecting domestic as well as wild birds. Asdescribed for helminth infections in mammals, A. galli infections in

dx.doi.org/10.1016/j.vaccine.2013.11.034


http://www.sciencedirect.com/science/journal/0264410X

http://www.elsevier.com/locate/vaccine

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hickens are associated with local T cell infiltrations in the intesti-al mucosa [7] as well as a Th2 polarized cytokine response [7–9] (&leidrup, unpublished observations). A systemic humoral immuneesponse also develops upon A. galli infection although serum anti-ody titres are non-persistent and do not appear to correlate withorm burden or egg excretion rates [7,10,11]. The adult parasite

esides in the lumen of the small intestine of the chicken andnfection is associated with impaired feed conversion and reduced

eight gain [12,13]. The prevalence is up to 100% in chicken flocksith outdoor access [14–17]. Thus, A. galli potentially represents a

erious problem if the infection hampers disease control throughaccination, e.g. in free range and organic chicken flocks.

Newcastle disease (ND) is a disease of significant economicmportance and hence controlled by vaccination in many countries

ith commercial poultry production [18]. Horning et al. [16] inves-igated the influence of A. galli infection on Newcastle disease virusNDV) vaccination in naturally parasite-infected indigenous chick-ns and found that dewormed NDV-vaccinated chickens showed

higher NDV-specific antibody response upon challenge with vir-lent velogenic NDV. However, neither cellular vaccine responsesor vaccine-induced protective immunity were addressed in the

ormer study. The purpose of this study was therefore to inves-igate cellular and humoral NDV vaccine responses and comparerotective immunity induced in A. galli-infected individuals asell as in non-parasitized controls using chickens from an MHC-

haracterized inbred line in a controlled experimental challengenfection.

. Materials and methods

.1. Animals

Chickens from line 133 kept at Aarhus University were used.ine 133 is of White Leghorn origin containing birds with the MHCaplotype B13. The experiment was initiated when the chickensere four weeks of age in order to avoid influence from maternal

ntibodies. Mixed gender was used. Water and commercial chickeneed were supplied ad libitum. The lighting period was 12 h daily,nd the chickens were subjected to a temperature of 21 ◦C.

All chickens used in the experiment were produced from MHC-haracterized parents. The MHC haplotypes of the offspring wereonfirmed by genotyping the LEI0258 microsatellite locus [19]y PCR-based fragment analysis. In brief, red blood cells fromeripheral blood were used as template for PCR using the Phu-ion Blood Direct PCR kit (Finnzymes, Espoo, Finland) accordingo the manufacturer’s instructions. Amplification by PCR and gelocumentation were performed as earlier described [20].

.2. Experimental outline

Chickens were wing-banded at hatch and divided into fourifferent experimental groups; (1) no A. galli infection, no ND vacci-ation (A−N−), (2) no A. galli infection, ND vaccination (A−N+), (3). galli infection, no ND vaccination (A+N−) and (4) A. galli infec-ion and ND vaccination (A+N+). Eight animals from each groupere used for blood sampling and oral swabs throughout the study.

even animals from each group were used for spleen and jejunumampling at each time-point.

The experimental A. galli infection (1750 infective A. galliggs/animal) was performed when the animals were four weeksf age (Day 0 PI). At 10 and 13 weeks of age, the chickens were ND-


accinated using Nobilis ND C2 (live attenuated strain Ulster C2,ntervet). Vaccination days were designated day 0 post-vaccination

and 2 (PV1 and PV2), respectively. The Ulster C2 strain is ansymptomatic enteric strain (genotype I) and was given nasally,

PRESSxxx (2013) xxx– xxx

>107.5 EID50/dose. During the first part of the experiment, vac-cinated and non-vaccinated controls were kept in separate unitsof the facility and within each unit, A. galli-infected chickens andnon-parasitized controls were placed in separate rooms. At 16weeks of age (12 weeks PI) chickens were transferred to four dif-ferent negative pressure isolators and exposed to NDV challengeinfection (CH), i.e. the chickens were infected by nasal inocula-tion of 200 �l LaSota virus (titer 109.5 ELD50/ml) diluted 1:100 inPBS. The virus was cultivated by passage in embryonated specifiedpathogen-free (SPF) hen eggs according to the 92/66/EEC councildirective (http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CONSLEG:1992L0066:20080903:EN:PDF). Titration of the viruswas done by inoculation of a 10-fold dilution series in four embry-onated chicken eggs for each step.

2.3. A. galli- and NDV-specific IgG ELISA

Blood samples were taken on day 0 PV1 and continuously everyweek throughout the experiment and serum was used for detectionof A. galli- and NDV-specific IgG (IgY) antibodies. The A. galli-specific IgG titres were measured as described by Norup et al. [11].Briefly, 96-well microtitre plates coated with A. galli soluble antigen(5 �g/ml) were incubated with serum samples, controls or stan-dards. Horseradish peroxidase-conjugated goat anti-chicken IgG(AAI29P, AbD Serotec, Oxford, UK) was used as detection antibodyand coloration was driven by tetramethylbenzidine. The resultswere recorded as the optical density (OD) at 450 nm with 650 nm asreference. A dilution series of a highly positive serum was used asstandard. The NDV-specific IgG ELISA was performed as previouslydescribed in Dalgaard et al. (2010), using the Flock Check* NDVELISA kit (Idexx Laboratories Westbrook, Maine, USA) following thekit manual. The antibody titres were calculated from a sample topositive ratio (S/P) as log10 titre = (1.09 × log10 S/P) + 3.36, the S/Pbeing calculated from negative control mean (NCx̄) and positivecontrol mean (PCx̄) as (sample mean − NCx̄/PCx̄ − NCx̄).

2.4. Faecal A. galli egg excretion

Faecal samples were obtained from all A. galli-infected chickensat day 0 PV1, at day 0 PV2 and at the day of the NDV challenge (day0 PCH). The faecal samples were examined for the presence of A.galli eggs using a modified McMaster counting technique [21,22]with a detection limit of 20 eggs per gram faeces (EPG).

2.5. Real-time quantitative RT-PCR–NDV genomes in oral swabs

Oral swabs were taken from eight animals from each of thefour experimental groups at days 0, 3 and 7 PV1 and PV2, respec-tively, and at days 0, 2, 4 and 7 post-CH (PCH). Sampled swabswere placed in PBS supplemented with penicillin (2000 U/ml),streptomycin (2 mg/ml), foetal calf serum (FCS, 5%) and kept at−20 ◦C for later analysis. Swab samples from the same groupwere pooled before RNA isolation from day 0 PV1, day 0 PV2 andday 0 PCH. From all other days RNA was isolated from individ-ual samples. The MagMAXTM 96 Viral RNA Isolation Kit (AppliedBiosystems/Ambion, Foster City, CA, USA) was used to isolate RNAfrom 45 �l swab or swab pool according to the manufacturer’sinstructions. RNA was eluted in 40 �l Elution Buffer from which5 �l was used as template for one-step qRT-PCR with Taqman®NDVReagents and Controls (Applied Biosystems). The qRT-PCR reactionwas performed on a ABI PRISM 7900HT instrument (Applied Biosys-


tems) programmed to cycle 10 min at 48 ◦C, 10 min at 95 ◦C, 40×(15 s at 95 ◦C and 45 s at 60 ◦C). The NDV-specific primers and probeincluded in the kit were designed to amplify several NDV strains,including LaSota and Ulster, as described in [23].


http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CONSLEG:1992L0066:20080903:EN:PDF

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.5.1. NDV recall antigen stimulation of PBMCPeripheral blood mononuclear cells (PBMC) were purified from

eparinized blood by Ficoll density gradient centrifugation. Inrief, the blood was diluted 1:2 with PBS and layered onto Ficoll-aqueTM PLUS, density 1.077 g/ml (Amersham Biosciences AB,ppsala, Sweden) before centrifugation at 400 × g for 25 min. TheBMC-containing interface as well as the supernatant above it wereubsequently transferred to new tubes and washed twice with PBSy centrifugation at 500 × g for 5 min. The PBMC were resuspended

n R5 medium (RPMI 1640 with 5% FCS) and the concentra-ion adjusted to 2 × 107 cells/ml. For antigen stimulation 50 �lells (1 × 106)/well were used in 96-well plates (Nunclon®Surface,unc, Roskilde, Denmark). The cells were stimulated with 50 �l of5 medium containing viral antigen or ConA. The final concentra-ions were 2 doses of NDV antigen (added day 1) or 10 mg/ml ofonA (added day 0). A negative control with medium alone waslso included. The NDV antigen was prepared as earlier described24] using an equal mixture of UV-inactivated commercial NDV vac-ines, i.e. Poulvac NDV (Ulster strain >106.0 EID50/dose, Fort Dodge,nimal Health Ltd., Southampton, UK) and ND C2 (C2 strain >107.5

ID50/dose, Intervet, Boxmeer, Netherlands). In total, the cells wereeft in culture for three days in 5% CO2 at 41 ◦C.

.5.2. Antibodies and flow cytometryAll monoclonal antibodies were obtained from Southern Biotech

Birmingham, Alabama, USA). Antibodies specific for chickenurface markers were: CD3-SPRD(CT-3), CD4-RPE(CT-4), CD8�-YTM5(3-298) and TCR��-FITC (TCR1). Propidium iodide wasbtained from Fluka BioChemika (Buchs, Switzerland). Titration ofll antibodies was done prior to the experiment in order to deter-ine the optimal staining concentrations. After three days, 50 �l of

he supernatants were removed from the cultured cells and EDTAas added to all samples in a final concentration of 2 mM in 50 �l5. The plates were left for 5 min on a gently rocking plate mixer,nd subsequently for 10 min in the CO2 incubator at 41 ◦C. Mono-lonal antibodies were then added in 50 �l FACS buffer (0.2% BSA,.2% sodium azide, 0.05% normal horse serum in PBS) and stain-

ng performed at 4 ◦C for 15 min followed by washing twice in50 �l FACS buffer with centrifugation at 600 × g for 5 min. Aftericking off the supernatant, the cells were finally resuspended in50 �l FACS buffer and analyzed by flow cytometry using a BD FACSantoTM (Beckton Dickinson, San Jose, CA) equipped with a 488 nmlue laser and a 633 nm red laser. Acquisition was performed with

fixed time of 30 s/sample and results analyzed using the FACSDivaoftware version 6.0 (BD Biosciences). T cell responses were quan-ified by determination of lymphoblast frequencies as originallyescribed in [25]. For the staining panel, single-stained compen-ation controls as well as negative fluorescence minus one (FMO)26] controls were used.

.5.3. Real-time quantitative RT-PCR-cytokine expressionCytokine mRNA expression was measured in spleen and

ejunum at day 14 PI, day 1 PV1 and day 1 PV2. Tissue samplesere immediately placed in RNAlater (Ambion, Austin, Texas, USA),

ept overnight at 4 ◦C and then at −20 ◦C until further processing.mounts of 7–15 mg tissue were homogenized on a TissueLyzer LT

Qiagen, Hilden, Germany), and RNA isolation was done using theucleoSpin 96 RNA kit (Macherey-Nagel) according to the manu-

acturer’s instructions. RNA quality was controlled on a 1% agaroseel and the RNA concentration and purity was determined using aanoDrop spectrophotometer (Saveen and Werner AB, Limhamn,weden). All samples were adjusted to the same total RNA concen-


ration. The RT-PCR was performed according to the manufacturer’snstructions for the TaqMan One Step Master Mix Reagent kitApplied Biosystems). In brief, per sample/determination a mix-ure of 5 �l of 2× MasterMix, 9 pmol of each primer, 2.5 pmol of

PRESSxxx (2013) xxx– xxx 3

the probe and 0.24 �l MultiScribe enzyme mixture added up to8 �l with DEPC water was made. Two �l of diluted RNA (320 ng)was added to 8 �l of the mixture and the qRT-PCR reaction wasperformed on a 7900HT instrument (Applied Biosystems) pro-grammed to cycle 30 min at 48 ◦C, 10 min at 95 ◦C, 40× (15 s at95 ◦C and 1 min at 60 ◦C). Sequences of Taqman primers and probesare shown in Table 1. Samples were run in duplicates and quan-tification of the mRNA expression was done using the RelativeStandard Curve method. A 5-fold dilution series prepared from sin-gle high-responding samples was used as standard. qRT-PCR datawere further validated using two-step qRT-PCR in a 48.48 DynamicArray Integrated Fluidic Circuits (Fluidigm Corporation, CA, USA)as previously described in [27]. All spleen samples were reana-lyzed using different sets of primers targeting the same five genesof interest and several validated internal reference genes.

2.5.4. Ethics statementLicense to conduct the animal experiment was obtained from

the Danish Ministry of Justice, Animal Experimentation Inspec-torate by Helle R. Juul-Madsen. The experiment was conductedaccording to the ethical guidelines

2.5.5. Statistical analysisDifferences in mean IgG titres, T cell responses and qPCR data

(NDV RNA genomes in oral swabs from day 0 to day 7 PCH andcytokine mRNA expression in tissue), between days, groups andwithin groups were analyzed by Analysis of Variance using a gen-eralized linear model (GLM) procedure in SAS software (SAS 9.2).Cytokine mRNA expression data were log-transformed to approachnormal distribution. Analyses were made comparing least of squaremeans (LS means). GLM procedures were described by the equa-tion:

Y = m + G + D + G x D + e

where Y is the observed value, � is the intercept, G is the group, Dis the day and e is the error.

EPG counts were analyzed by Chi-Square tests, and NDV RNAexpression data, from day 0 PV1 to day 7 PV2 before challenge, wasanalyzed by a t test. The differences were considered statisticallysignificant at P < 0.05. All data presented in figures and tables areoriginal and presented as non-transformed data.

3. Results

3.1. A. galli-specific serum IgG and faecal A. galli egg excretion

All chickens in the A. galli-inoculated groups (3 and 4) respondedwith A. galli-specific serum IgG titres from day 0 PV1 (6 weeks PI)and continuously throughout the experiment. Most faecal samplescollected from the A. galli-infected groups tested positive for A. gallieggs on one or more of the three time-points; day 0 PV1 (6 weeksPI), day 0 PV2 (9 weeks PI) and day 0 PCH (16 weeks PI). No sig-nificant differences in A. galli-specific IgG titres or EPG levels werefound between the two infected groups at any time-point (datanot shown). None of the chickens in the A. galli-free groups (1 and2) tested sero-positive or EPG positive at any time-point duringthe experiment (Data not shown). The variation in EPG betweenindividuals was large and no correlations between EPG and A. galliserum titres or NDV serum titres were found.

3.2. NDV-specific serum IgG


The NDV-specific serum titres in the vaccinated groups (2 and4) increased significantly from week 2 PV1 (Fig. 1). Considering theentire experimental period, significantly higher NDV titres were


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Table 1Real-time quantitative RT-PCR probes and primers.

RNA target Probe/primer sequences Exon boundary Accession No.a

IFN-� Probe5′-(FAM)-TGGCCAAGCTCCCGATGAACGA-(TAMRA)-3′ 3/4 Y07922Forward 5′-GTGAAGAAGGTGAAAGATATC ATGGA-3′

Reverse 5′-GCTTTGCGCTGGATTCTC A-3′

IL-4 Probe 5′-(FAM)-AGC AGCACCTCCCTCAAGGCACC-(TAMRA)-3′ 3/4 AJ621735Forward 5′-AAC ATGCGTC AGCTCCTGAAT-3′

Reverse 5′-TCTGCTAGGAACTTCTCCATTGAA-3′

IL-13 Probe 5′-(FAM)-CATTGCAAGGGACCTGCACTCCTCTG-(TAMRA)-3′ 1/2 AJ621735Forward 5′-CACCCAGGGCATCCAGAA-3′

Reverse 5′-TCCGATCCTTGAAAGCC ACTT-3′

IL-10 Probe 5′-(FAM)-CGACGATGCGGCGCTGTCA-(TAMRA)-3′ 3/4 AJ621614Forward 5′-CATGCTGCTGGGCCTGAA-3′

Reverse 5′-CGTCTCCTTGATCTGCTTGATG-3′

TGF-�4 Probe 5′-(FAM)-ACCCAAAGGTTATATGGCCAACTTCTGCAT-(TAMRA)-3′ 6/7 M31160Forward 5′-AGGATCTGC AGTGGAGTGGAT-3′

Reverse 5′-CCCCGGGTTGTGTTGGT-3′

Sequences of the oligonucleotide primers and probes used in quantitative RT-PCR are identical to primers and probes used in Avery et al. and Rothwell et al. [43,50].a Refers to the genomic DNA sequence.

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significantly lower in group 3 as compared to group 1. In Table 2the frequency of NDV-positive animals PCH is shown.

Table 2Frequencies of NDV positive animals, determined by detection of genomic RNA inoral swaps at day 2, 4 and 7 post NDV challenge.

Day 2 PCH Day 4 PCH Day 7 PCH

ig. 1. NDV-specific IgG titres in serum from the different experimental groups; (1accine (A+N−) and (4) A. galli and ND vaccine (A+N+). Data are shown as mean ± S

ound in non-parasitized chickens from group 2 (P = 0.0477) as com-ared to chickens in the A. galli-infected group 4. Thus, at week 2V1, week 0 PV2 and week 1 PV2, NDV titres were significantlyigher in group 2 than in group 4. At 2 weeks PCH, the NDV titresere increased in both vaccinated groups (2 and 4) when compared

o the day of challenge, but no significant difference was observedetween the groups. In the non-vaccinated groups, seropositivityas observed at week 1 PCH and at this time-point the titre was

lightly higher in A. galli-infected chickens (group 3) as comparedo non-parasitized controls (group 1) (P = 0.0511). By 2 weeks PCH,ll experimental groups had identical NDV-specific IgG titers.

.3. NDV load in oral swabs

NDV genomes/viral load in oral swabs were determined by realime qRT-PCR using primers specific for both the Ulster C2 vac-


ine strain and the LaSota challenge strain. The viral genomes wereetectable in groups 2 and 4 from day 3 PV1 (Fig. 2) and group 4howed a slightly higher mean viral load than group 2 (not signif-cantly). From day 7 PV1 NDV genomes were no longer detectable

. galli, no ND vaccine (A−N−), (2) no A. galli, ND vaccine (A−N+), (3) A. galli, no ND8). *P < 0.05.

but after NDV challenge all groups were positive for viral genomes.Thus, the NDV-vaccinated groups (2 and 4) had significantly fewerNDV genomes than the non-vaccinated groups (1 and 3) on days2 and 4 PCH. At day 2 PCH, group 4 was significantly differentfrom zero which was not the case for group 2. By day 7 PCH, NDVgenomes were no longer detectable in oral swabs from groups 2and 4, and the viral load had decreased significantly in both non-vaccinated groups (1 and 3). The viral load at this time-point was


Group 1: A−N− 8/8 6/8 6/8Group 2: A−N+ 3/8 2/8 0/8Group 3: A+N− 8/8 7/8 3/8Group 4: A+N+ 6/8 4/8 0/8




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.4. NDV-specific T cells in peripheral blood

The presence of NDV-specific T cells in peripheral blood wasssessed at week 1 PV1, week 3 PV1, week 1 PV2 and week 3V2 by ex vivo recall stimulation and flow cytometric lymphoblastnalyses. The gating strategy is shown in Fig. 3a. The lymphoblastesponse was analyzed in two different CD3+ subsets: CD8veells (TCR1-CD8�+) and CD4ve cells (TCR1-CD4+). No significantifferences in blast frequency in the medium controls or ConA-timulated cells were seen between the four treatment groups (dataot shown). Blast frequencies upon stimulation with NDV antigenre shown in Fig. 3b and c. Significant differences were found 3eeks PV1 for both T cell subsets. Vaccinated chickens in group

showed a higher NDV-specific response as compared to naïvehickens in group 1 (and 3). In vaccinated chickens from group 4o NDV-specific response was detected as lymphoblast frequen-ies were not higher than the background seen in non-vaccinatedhickens in group 3.

.5. Cytokine mRNA expression

Cytokine expression was measured at day 14 PI, day 1 PV1 anday 1 PV2, for the Th1 cytokine IFN-�, the Th2 cytokines IL-4 and

L-13 and the regulatory cytokines IL-10 and TGF-�4 in spleen andejunum (Fig. 4). Before vaccination, at day 14 PI, only two groupsxisted A. galli-infected (designated 3) and A. galli-uninfecteddesignated 1) from which seven random animals were tested.

At day 14 PI no significant differences between A. galli-infectednd uninfected animals were found in the spleen for IFN-�. In con-rast, significant differences were found between groups at day

PV1 and day 1 PV2. Thus, by day 1 PV1 a significantly lowerxpression of IFN-� (2-fold) was found in A. galli-infected and vacci-ated chickens (group 4) as compared to non-parasitized controlsgroups 1 and 2). No vaccine-induced difference in IFN-� mRNAxpression was observed (i.e. 1 vs. 2 and 3 vs. 4). By day 1 PV2he non-parasitized/non-vaccinated group (group 1) showed sig-ificantly higher IFN-� mRNA expression in spleen than the threether groups. In the jejunum, no difference in IFN-� mRNA expres-


ion was found at day 14 PI, but significant differences were foundt the other two time-points. Thus, at both time-points PV the A.alli-infected groups (3 and 4) had a lower expression (1.8-fold)han the non-parasitized groups (1 and 2).

tal groups; (1) no A. galli, no ND vaccine (A−N−), (2) no A. galli, ND vaccine (A−N+),as mean ± SE (n = 8). *3 /= 1, P < 0.05.

At day 14 PI, IL-4 expression was significantly higher (2.5-fold) in the jejunum of A. galli-infected animals as compared tonon-parasitized controls. At the two time-points PV, IL-4 mRNAexpression differed significantly in the spleen at day 1 PV2 and thisdifference was vaccine-induced repression of IL-4 expression (3.3-fold) only found in the non-parasitized groups (2 relative to 1) andnot in the A. galli-infected groups (4 relative to 3).

IL-13 mRNA expression at day 14 PI was significantly higher inboth spleen and jejunum from A. galli-infected chickens as com-pared to non-parasitized controls, with fold changes of 7.5 and3842, respectively. At day 1 PV1 and day 1 PV2 the relative expres-sion of IL-13 decreased dramatically compared to day 14 PI. Inthe spleen, significantly higher (2.5–5.3-fold) IL-13 expression wasfound in A. galli-infected NDV-vaccinated animals (group 4) at day1 PV1 compared to the three other groups. Although the expres-sion appeared low in the jejunum, the A. galli-infected groups (3and 4) showed significantly higher IL-13 mRNA expression thanthe non-parasitized groups (group 1 and 2) at both time-points PV.

IL-10 mRNA expression did not differ significantly betweenA. galli-infected and non-infected animals at day 14 PI in eitherspleen or jejunum. At day 1 PV1 and day 1 PV2 significantlylower (15–38-fold) mRNA expression of IL-10 in the spleen wasfound in the A. galli-infected groups (3 and 4) compared tonon-parasitized control groups (1 and 2). In addition, a vaccine-induced decrease in IL-10 expression (1.6-fold) was found betweengroups 1 and 2 at day 1 PV1, but not between groups 3 and4. In the jejunum, significantly lower IL-10 expression was alsofound in groups 3 and 4 compared to groups 1 and 2 at day1 PV1 (5.8–15.6-fold). By day 1 PV2 vaccine-induced higher IL-10 mRNA expression was found in the non-parasitized groups(1 and 2) which was not seen for the A. galli-infected groups(3 and 4).

Significant differences in TGF-�4 expression between infectedand non-infected animals were found on day 14 PI in both tissues asA. galli-infected animals showed significantly lower (1.9–2.9-fold)expression as compared to non-parasitized controls. In the jejunumat day 1 PV1, significantly lower TGF-�4 mRNA expression was fur-thermore found in A. galli-infected and vaccinated animals (group4) compared to non-parasitized NDV vaccinated animals (group 2).


Results from the two-step qRT-PCR data analysis generally vali-dated the above findings very well. However, we were not able toshow the same degree of TGF-�4 down-regulation at day 14 PI inthe spleen of A. galli-infected animals.




6 J. Pleidrup et al. / Vaccine xxx (2013) xxx– xxx

Fig. 3. Peripheral NDV-specific T cells assessed by flow cytometry. (a) Gating strategy for analyses of CD4 cells (TCR1-CD4+) and CD8 cells (TCR-CD8+) (Con A example).Lymphoblasts were identified from their FSC/SSC profiles and frequencies recorded within each T cell subset. T cell lymphoblast frequencies induced by ex vivo NDV recalls e (A−A in (c)s

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timulation in the four different experimental groups; (1) no A. galli, no ND vaccin. galli and ND vaccine (A+N+). CD8 cells are shown in (b) and CD4 cells are shownubset indicate significant differences (P < 0.05).

. Discussion


In the present study, A. galli infection influenced the humoralaccine responses in chickens exposed to the parasite prior to NDaccination (Fig. 1). Differences were observed after ND vaccina-ion, but not after NDV challenge. This is in contrast to a former

N−), (2) no A. galli, ND vaccine (A−N+), (3) A. galli, no ND vaccine (A+N−) and (4). Data are expressed as mean ± SE (n = 8). Different superscript letters within each

study showing reduced humoral antibody response to NDV chal-lenge of ND-vaccinated A. galli-infected chickens [16]. However,


the former study compared naturally A. galli-infected indigenouschickens with and without deworming whereas the present studycompares experimentally infected versus non-infected chickensfrom an inbred MHC-characterized line. Genetics and possibly MHC


Please cite this article in press as: Pleidrup J, et al. Ascaridia galli infection influences the development of both humoral and cell-mediatedimmunity after Newcastle Disease vaccination in chickens. Vaccine (2013), http://dx.doi.org/10.1016/j.vaccine.2013.11.034



J. Pleidrup et al. / Vaccine xxx (2013) xxx– xxx 7

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Fig. 4. Relative IFN-� (a), IL-4 (b), IL-13 (c), TGF-�4 (d), IL-10 (e) cytokine mRNA expression measured by qRT-PCR in jejunum and spleen. Before ND vaccination, only twogroups existed, i.e. non-A. galli-infected (designated 1) and A. galli-infected chickens (designated 3). Immediately prior to vaccination, chickens were divided into the fourdifferent experimental groups; (1) no A. galli, no ND vaccine (A−N−), (2) no A. galli, ND vaccine (A−N+), (3) A. galli, no ND vaccine (A+N−) and (4) A. galli and ND vaccine(A+N+). Data are shown as mean ± SE (n = 7). Different superscript letters within each gene indicate significant differences (P < 0.05).


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aplotype were earlier shown to influence A. galli serum titres andgg excretion [11] and the chicken line used in the current study isnown to exhibit an intermediate response when comparing eightifferent MHC lines (unpublished observations). Furthermore, theorning study was based on vaccination with live attenuatedaSota (lentogenic) virus intra-ocularly and challenge with NDVrom a field outbreak (velogenic) by contact with infected seederhickens. The present study used a live attenuated NDV Ulster stainasymptomatic enteric) as nasal vaccine and LaSota (lentogenic) asasal challenge strain. Thus the different vaccine/challenge mod-ls may contribute to the differences in humoral immune responsebserved between the two studies.

In the present study, a slightly higher (not significantly) viraload at day 2 PCH and a higher frequency of NDV-positive animalsn days 2 and 4 PCH were found in the A. galli-infected animalsFig. 2; Table 2), indicating a potential impact of A. galli on vaccine-nduced disease protection. Interestingly, in the non-vaccinatednimals, the virus was cleared faster in the A. galli-infected ani-als on day 7 PCH, for which we have no explanation. It may be

ypothesized that NDV challenge in combination with A. galli infec-ion has changed the time course of the immune response directedgainst the virus. In support of this theory, the NDV antibody titreas higher in the A. galli-infected animals at the same time-point

although not significantly higher than the non-A. galli-infectedroup, (P = 0.0511)).

In addition to humoral immunity, which indeed plays a rolen protective immunity against NDV, commercial live NDV vac-ines can also induce cell-mediated immunity [28,29]. NDV-specific

cells play a role in viral clearance upon infection and havearlier been demonstrated in peripheral blood and spleen from ND-accinated chickens [24,30,31]. In the present experiment, CD4+nd CD8+ T cell blastogenesis induced by NDV-recall antigen wasetected in ND-vaccinated chickens three weeks PV1, but not in A.alli-infected NDV-vaccinated chickens (Fig. 3b and c) indicating anmpaired cellular immune response caused by A. galli infection. Thiss in good agreement with earlier murine and human studies show-ng that helminth infections decrease virus-specific CD8+ cytotoxic

cell responses and impair proliferation of PBMC in response toecall antigens [32–34]. In the present study it was not possible toetect blastogenesis of NDV-specific T cells in peripheral blood afterhe 2nd vaccination, as groups 2 and 4 did not differ from group 1.his is in contrast to the earlier results where T cell proliferation wasetected upon PBMC stimulation with NDV recall antigen at week

post-secondary vaccination [24]. However, although the chickensere from the same MHC-inbred line in the two experiments, age

t vaccination as well as vaccine strain differed between the twoxperiments.

Only a few differences in cytokine mRNA expression caused byDV vaccine alone (group 1 vs 2) or in combination with A. galli

nfection (group 2 vs 4) were observed in this study. However, sev-ral differences caused by A. galli infection alone (group 1 vs 3)ere observed. Thus, in the present study A. galli-infected chickens

Fig. 4 group 3) showed reduced expression of the Th1 signatureytokine IFN-� as compared to controls (group 1) in the spleen andejunum six and nine weeks post-A. galli infection, but not at 14ays PI. This is in contrast to earlier findings by Degen et al. [8] whoeported decreased relative cytokine mRNA ratios (infected/non-nfected) for IFN-� in both spleen and ileal tissue (caecal tonsils,ersonal communication) in layer chickens already at two weeksost-infection. Schwarz et al. [7] on the other hand found elevated

FN-� cytokine mRNA expression in jejunal tissue two to threeeeks after infection with A. galli which may be explained by the


elatively low dose (250) of embryonated A. galli eggs used in theirtudy. In the current study and the Degen study, 1750 and 1000ggs were used, respectively. Earlier reports do suggest that onsetnd length of the larvae histotrophic phase depend on infection


dose, and furthermore the outcome of infection may be influencedby host genetics [11,35,36].

In the present study, the cytokine mRNA analyses of spleen andjejunum clearly showed elevated expression of the Th2 signaturecytokines IL-4 and IL-13 at day 14 post-A. galli infection (compar-ison of groups 1 and 3, Fig. 4) which is in agreement with resultspublished earlier [7–9]. Interestingly, no differences between con-trols (group 1) and A. galli-infected chickens (group 3) in IL-4expression in the jejunum or in the spleen were observed at sixand nine weeks PI, and only slight differences were observed inIL-13 expression with much lower fold changes between controlsand A. galli-infected chickens than were seen day 14 PI, whichis in good agreement with earlier reports [7]. Schwarz et al. [7]also reported A. galli-induced influx of both �� (including CD4+vecells) and �� T cells in the jejunal mucosa at week 2 PI but thelymphocyte infiltration declined at week 3 and 6 PI and conse-quently it may be hypothesized that reduced expression of IL-4and IL-13 in the jejunum coincides with declined infiltration ofTh2 cells. The A. galli life cycle should also be taken into accountas day 14 PI is within the larvae histotrophic phase and at weeks6 and 9 PI adult worms have settled in the lumen of the smallintestine [37].

In human and murine helminth infections it is evident thatparasite immunomodulation is a key player in survival strate-gies and that successful chronic infection requires down-regulationof host Th2 responsiveness [38,39]. From our results it may behypothesized that the same is true for helminth infections inchickens as Th2 responses weakened after development of adultworms. The survival strategy of helminth parasites is largelybased on immunoregulation by excretory-secretory (ES) productsthrough mechanisms involving regulatory T cells combined withTh2 anergy and exhaustion [39]. Interestingly, drug-induced clear-ance of helminths reduces Foxp3+ Treg numbers [3,40] and inmouse models Th2 immunity can be rescued by depletion of Tregs[41]. A Foxp3 orthologue has not yet been identified in the chicken,but thymic CD4+CD25+T cells were characterized as counterpartsof mammalian natural Tregs producing IL-10 and TGF-� with sup-pressive properties of T cell proliferation in vitro [42].

IL-10 has a conserved function in the chicken acting as an anti-inflammatory cytokine with the potential to down-regulate IFN-�-induced responses [43].

The avian TGF-� gene-family includes: TGF-�2, TGF-�3 andTGF-�4, of which the latter is the chicken orthologue of mam-malian TGF-�1 [44,45]. Indeed, chicken TGF-�4 acts as ananti-inflammatory cytokine [46,47]. Using another chicken para-site infection model (Eimeria spp.), Rothwell et al. [43] reported anincreased expression of IL-10 in infected susceptible chickens andSelvaraj [48] reported increased frequencies of CD4+CD25+ cells.In the present study we did not observe increased IL-10 or TGF-�4expression in A. galli-infected chickens. On the contrary, TGF-�4expression was down-regulated early (14 day PI and 6 weeks PI)and IL-10 later in the course of the A. galli infection (week 6 PI andweek 9 PI). Indeed, cells other than Tregs produce IL-10 and TGF-�4 and constitutive expression in the intestine of healthy chickenswas reported earlier [43,49] In order to determine if Tregs play arole in A. galli infections, cytokine responses should be addressedat the single cell level.

An NDV vaccine-induced decrease in expression was found atday 1 PV2 for IFN-� and IL-4 in the spleen of parasite-free chick-ens, but not in parasitized chickens. IL-10 expression decreased atday 1PV1 (spleen) and increased at day 1PV2 (jejunum) upon vac-cination of parasite-free chickens, but not in parasitized chickens.


Reports describing which cytokine mRNA profiles correlate withNDV vaccine-induced protection are needed in order to elucidate ifthe described differences are involved in reduced vaccine efficacyby helminth infections.


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In conclusion, A. galli infection in chickens influenced the devel-pment of both humoral and cell-mediated immune responsesnduced by a live attenuated commercial NDV vaccine, suggest-ng strong immunomodulatory potential for this parasite. In theresent study, NDV genomes persisted in the oral cavity for alightly longer period of time in A. galli-infected vaccinees as com-ared to non-parasitized vaccinees. However, more work is needed

n order to prove that vaccine-induced protective immunity iseduced in A. galli-infected chickens.

onflicts of interest statement

The authors declare to have no conflicts of interest.

cknowledgments

The authors wish to acknowledge financial support from thehe Danish Council for Strategic Research, Aarhus University andhe European Union Seventh Framework Network of Animal Dis-ase Infectiology Research Facilities (NADIR; reference numberP7-228394). Pete Kaiser and Lisa Rothwell are thanked for fruitfulomments and support, Lene Rosborg Dal and Helle Handll for tech-ical assistance, Martin Moerck Mortensen for graphic assistancend Karin V. Østergaard for proof reading of the manuscript.

eferences

[1] Cooper PJ, Chico M, Sandoval C, et al. Human infection with Ascaris lum-bricoides is associated with suppression of the interleukin-2 response torecombinant cholera toxin B subunit following vaccination with the live oralcholera vaccine CVD 103-HgR. Infect Immun 2001;69(March (3)):1574–80.

[2] Elias D, Wolday D, Akuffo H, Petros B, Bronner U, Britton S. Effect of deworm-ing on human T cell responses to mycobacterial antigens in helminth-exposedindividuals before and after bacille Calmette-Guerin (BCG) vaccination. ClinExp Immunol 2001;123(February (2)):219–25.

[3] Wammes LJ, Hamid F, Wiria AE, et al. Regulatory T cells in human geohelminthinfection suppress immune responses to BCG and Plasmodium falciparum. EurJ Immunol 2010;40(February (2)):437–42.

[4] Cooper PJ, Espinel I, Paredes W, Guderian RH, Nutman TB. Impairedtetanus-specific cellular and humoral responses following tetanus vaccina-tion in human onchocerciasis: a possible role for interleukin-10. J Infect Dis1998;178(October (4)):1133–8.

[5] Nookala S, Srinivasan S, Kaliraj P, Narayanan RB, Nutman TB. Impairment oftetanus-specific cellular and humoral responses following tetanus vaccinationin human lymphatic filariasis. Infect Immun 2004;72(May (5)):2598–604.

[6] Steenhard NR, Jungersen G, Kokotovic B, et al. Ascaris suum infection nega-tively affects the response to a Mycoplasma hyopneumoniae vaccination andsubsequent challenge infection in pigs. Vaccine 2009;27(August (37)):5161–9.

[7] Schwarz A, Gauly M, Abel H, et al. Immunopathogenesis of Ascaridia galli infec-tion in layer chicken. Dev Comp Immunol 2011;35(July (7)):774–84.

[8] Degen WG, Daal N, Rothwell L, Kaiser P, Schijns VE. Th1/Th2 polarizationby viral and helminth infection in birds. Vet Microbiol 2005;105(February(3–4)):163–7.

[9] Kaiser P. The avian immune genome—a glass half-full or half-empty? CytogenetGenome Res 2007;117(1–4):221–30.

10] Marcos-Atxutegi C, Gandolfi B, Aranguena T, Sepulveda R, Arevalo M, SimonF. Antibody and inflammatory responses in laying hens with experimentalprimary infections of Ascaridia galli. Vet Parasitol 2009;161(April (1–2)):69–75.

11] Norup LR, Dalgaard TS, Pleidrup J, et al. Comparison of parasite-specificimmunoglobulin levels in two chicken lines during sustained infection withAscaridia galli. Vet Parasitol 2013;191(January (1–2)):187–90.

12] Chadfield M, Permin A, Nansen P, Bisgaard M. Investigation of the parasiticnematode Ascaridia galli (Shrank 1788) as a potential vector for Salmonellaenterica dissemination in poultry. Parasitol Res 2001;87(April (4)):317–25.

13] Skallerup P, Luna LA, Johansen MV, Kyvsgaard NC. The impact of naturalhelminth infections and supplementary protein on growth performance offree-range chickens on smallholder farms in El Sauce, Nicaragua. Prev Vet Med2005;69(July (3–4)):229–44.

14] Das G, Kaufmann F, Abel H, Gauly M. Effect of extra dietary lysine in Ascaridiagalli-infected grower layers. Vet Parasitol 2010;170(June (3–4)):238–43.

15] Gauly M, Duss C, Erhardt G. Influence of Ascaridia galli infections andanthelmintic treatments on the behaviour and social ranks of laying hens (Gal-


lus gallus domesticus). Vet Parasitol 2007;146(May (3–4)):271–80.16] Horning G, Rasmussen S, Permin A, Bisgaard M. Investigations on the influ-

ence of helminth parasites on vaccination of chickens against Newcastledisease virus under village conditions. Trop Anim Health Prod 2003;35(October(5)):415–24.

[

PRESSxxx (2013) xxx– xxx 9

17] Permin A, Bisgaard M, Frandsen F, Pearman M, Kold J, Nansen P. Prevalence ofgastrointestinal helminths in different poultry production systems. Br Poult Sci1999;40(September (4)):439–43.

18] Alexander DJ. Newcastle disease in the European Union 2000 to 2009. AvianPathol 2011;40(December (6)):547–58.

19] McConnell SK, Dawson DA, Wardle A, Burke T. The isolation and mappingof 19 tetranucleotide microsatellite markers in the chicken. Anim Genet1999;30(June (3)):183–9.

20] Dalgaard TS, Vitved L, Skjodt K, et al. Molecular characterization of majorhistocompatibility complex class I (B-F) mRNA variants from chickens dif-fering in resistance to Marek’s disease. Scand J Immunol 2005;62(September(3)):259–70.

21] Henriksen SA, Aagaard K. [A simple flotation and McMaster method (author’stransl)]. Nord Vet Med 1976;28(July (7–8)):392–7.

22] Permin A, Bojesen M, Nansen P, Bisgaard M, Frandsen F, Pearman M. Ascaridiagalli populations in chickens following single infections with different doselevels. Parasitol Res 1997;83(6):614–7.

23] Wise MG, Suarez DL, Seal BS, et al. Development of a real-time reverse-transcription PCR for detection of newcastle disease virus RNA in clinicalsamples. J Clin Microbiol 2004;42(January (1)):329–38.

24] Dalgaard TS, Norup LR, Pedersen AR, Handberg KJ, Jorgensen PH, Juul-MadsenHR. Flow cytometric assessment of chicken T cell-mediated immune responsesafter Newcastle disease virus vaccination and challenge. Vaccine 2010;28(June(28)):4506–14.

25] Gaines H, Andersson L, Biberfeld G. A new method for measuring lymphopro-liferation at the single-cell level in whole blood cultures by flow cytometry. JImmunol Methods 1996;195(September (1–2)):63–72.

26] De Rosa SC, Brenchley JM, Roederer M. Beyond six colors: a new era in flowcytometry. Nat Med 2003;9(January (1)):112–7.

27] Skovgaard K, Cirera S, Vasby D, et al. Expression of innate immune genes,proteins and microRNAs in lung tissue of pigs infected experimentally withinfluenza virus (H1N2). Innate Immun 2013;(February).

28] Reynolds DL, Maraqa AD. Protective immunity against Newcastle disease: therole of cell-mediated immunity. Avian Dis 2000;44(January (1)):145–54.

29] Russell PH, Dwivedi PN, Davison TF. The effects of cyclosporin A and cyclophos-phamide on the populations of B and T cells and virus in the Harderian glandof chickens vaccinated with the Hitchner B1 strain of Newcastle disease virus.Vet Immunol Immunopathol 1997;60(December (1–2)):171–85.

30] Rauw F, Gardin Y, Palya V, et al. Humoral, cell-mediated and mucosal immu-nity induced by oculo-nasal vaccination of one-day-old SPF and conventionallayer chicks with two different live Newcastle disease vaccines. Vaccine2009;27(June (27)):3631–42.

31] Rauw F, Gardin Y, Palya V, et al. Improved vaccination against Newcastle diseaseby an in ovo recombinant HVT-ND combined with an adjuvanted live vaccineat day-old. Vaccine 2010;28(January (3)):823–33.

32] Actor JK, Shirai M, Kullberg MC, Buller RM, Sher A, Berzofsky JA. Helminthinfection results in decreased virus-specific CD8+ cytotoxic T-cell and Th1cytokine responses as well as delayed virus clearance. Proc Natl Acad Sci US A 1993;90(February (3)):948–52.

33] Borkow G, Bentwich Z. Chronic parasite infections cause immune changes thatcould affect successful vaccination. Trends Parasitol 2008;24(June (6)):243–5.

34] Schierack P, Lucius R, Sonnenburg B, Schilling K, Hartmann S. Parasite-specificimmunomodulatory functions of filarial cystatin. Infect Immun 2003;71(May(5)):2422–9.

35] Herd RP, McNaught DJ. Arrested development and the histotropic phase ofAscaridia galli in the chicken. Int J Parasitol 1975;5(August (4)):401–6.

36] Kaufmann F, Das G, Preisinger R, Schmutz M, Konig S, Gauly M. Genetic resis-tance to natural helminth infections in two chicken layer lines. Vet Parasitol2011;176(March (2–3)):250–7.

37] Katakam KK, Nejsum P, Kyvsgaard NC, Jorgensen CB, Thamsborg SM. Molecularand parasitological tools for the study of Ascaridia galli population dynamicsin chickens. Avian Pathol 2010;39(April (2)):81–5.

38] Maizels RM, Balic A, Gomez-Escobar N, Nair M, Taylor MD, Allen JE.Helminth parasites—masters of regulation. Immunol Rev 2004;201(October(89–116)):89–116.

39] Taylor MD, van der Werf N, Maizels RM. T cells in helminth infection: theregulators and the regulated. Trends Immunol 2012;33(April (4)):181–9.

40] Watanabe K, Mwinzi PN, Black CL, et al. T regulatory cell levels decrease inpeople infected with Schistosoma mansoni on effective treatment. Am J TropMed Hyg 2007;77(October (4)):676–82.

41] Blankenhaus B, Klemm U, Eschbach ML, et al. Strongyloides ratti infectioninduces expansion of Foxp3+ regulatory T cells that interfere with immuneresponse and parasite clearance in BALB/c mice. J Immunol 2011;186(April(7)):4295–305.

42] Shanmugasundaram R, Regulatory Selvaraj RK. T cell properties of chickenCD4 + CD25+ cells. J Immunol 2011;186(February (4)):1997–2002.

43] Rothwell L, Young JR, Zoorob R, et al. Cloning and characterization of chickenIL-10 and its role in the immune response to Eimeria maxima. J Immunol2004;173(August (4)):2675–82.

44] Jakowlew SB, Mathias A, Lillehoj HS. Transforming growth factor-beta iso-forms in the developing chicken intestine and spleen: increase in transforming


growth factor-beta 4 with coccidia infection. Vet Immunol Immunopathol1997;55(March (4)):321–39.

45] Pan H, Cloning Halper J. expression, and characterization of chicken trans-forming growth factor beta 4. Biochem Biophys Res Commun 2003;303(March(1)):24–30.


http://refhub.elsevier.com/S0264-410X(13)01551-X/sbref0005










































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































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[50] Avery S, Rothwell L, Degen WD, et al. Characterization of the first nonmam-


0 J. Pleidrup et al. / Va

46] Kogut MH, Rothwell L, Kaiser P. Differential regulation of cytokine geneexpression by avian heterophils during receptor-mediated phagocytosis ofopsonized and nonopsonized Salmonella enteritidis. J Interferon Cytokine Res2003;23(June (6)):319–27.


47] Swaggerty CL, Kogut MH, Ferro PJ, Rothwell L, Pevzner IY, Kaiser P. Differentialcytokine mRNA expression in heterophils isolated from Salmonella-resistantand -susceptible chickens. Immunology 2004;113(September (1)):139–48.

48] Selvaraj RK. Avian CD4CD25 regulatory T cells: Properties and therapeuticapplications. Dev Comp Immunol 2013;13(May):10.


49] Lammers A, Wieland WH, Kruijt L, et al. Successive immunoglobulin andcytokine expression in the small intestine of juvenile chicken. Dev CompImmunol 2010;34(December (12)):1254–62.


malian T2 cytokine gene cluster: the cluster contains functional single-copygenes for IL-3, IL-4, IL-13, and GM-CSF, a gene for IL-5 that appears to be a pseu-dogene, and a gene encoding another cytokinelike transcript, KK34. J InterferonCytokine Res 2004;24(October (10)):600–10.


































































































































































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