Effect of Eimeria acervulina infection history on the immune response and transmission in broilers.

Abstract

Heterogeneity in exposure to Eimeria spp. of chickens in a flock will result in differences between individual birds in oocyst output and acquired immunity, which subsequently affects transmission of the parasite in the population. The aim of this study was to quantify effects of previous infection of broilers with Eimeria acervulina on immune responses, oocyst output and transmission. A transmission experiment was carried out with pair-wise housed broilers, that differed in infection history. This "infection history" was achieved by establishment of a primary infection by inoculation of birds with 50,000 sporulated E. acervulina oocysts at day 6 of age ("primed"); the other birds did not receive a primary infection ("naïve"). The actual transmission experiment started at day 24 of age: one bird (I) was inoculated with 50,000 sporulated oocysts and was housed together with a non-inoculated contact bird (C). Oocyst excretion and parameters describing transmission, i.e. the number of infected C birds and time passed before start of excretion of C birds, were determined from day 28 to day 50 for six pairs of four different combinations of I and C birds (I-C): naïve-naïve, naïve-primed, primed-naïve and primed-primed. Immune parameters, CD4(+), CD8(+), αβTCR(+) and γδTCR(+) T cells and macrophages in duodenum, were determined in an additional 25 non-primed, non-inoculated control birds, and in the naïve-naïve and naïve-primed groups, each group consisting of 25 pairs. Although the numbers of CD4(+) T cells and γδTCR(+) T cells increased after primary infection, none of the immunological cell types provided an indication of differences in infectivity, susceptibility or transmission between birds. Oocyst output was significantly reduced in primed I and C birds. Transmission was reduced most in the primed-primed group, but nonetheless transmission occurred in all groups. This study also showed that acquired immunity significantly reduced oocyst output after inoculation and contact-infection, but not sufficiently to prevent transmission to contact-exposed birds.

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Veterinary Parasitology 173 (2010) 184–192
Contents lists available at ScienceDirect
Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar
Effect of Eimeria acervulina infection history on the immune response
and transmission in broilers
F.C. Velkersa,b,∗,1, W.J.C. Swinkelsa,1, J.M.J. Rebelc, A. Boumaa, A.J.J.M. Daemena,
D. Klinkenberga, W.J.A. Boersmac, J.A. Stegemana, M.C.M. de Jongb, J.A.P. Heesterbeeka
aDepartment of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80151, 3508 TD Utrecht, The Netherlands
bQuantitative Veterinary Epidemiology Group, Wageningen Institute of Animal Sciences, Wageningen University, P.O. Box 338, 6700 AH Wageningen, The
Netherlands
cCentral Veterinary Institute, P.O. Box 65, 8200 AB Lelystad, The Netherlands
a r t i c l ei n f o
Article history:
Received 4 December 2009
Received in revised form 8 July 2010
Accepted 15 July 2010
Keywords:
Eimeria acervulina
Broilers
Transmission
Immunity
Infection dynamics
Immuno-epidemiology
a b s t r a c t
Heterogeneity in exposure to Eimeria spp. of chickens in a flock will result in differences
between individual birds in oocyst output and acquired immunity, which subsequently
affects transmission of the parasite in the population. The aim of this study was to quan-
tify effects of previous infection of broilers with Eimeria acervulina on immune responses,
oocyst output and transmission. A transmission experiment was carried out with pair-wise
housed broilers, that differed in infection history. This “infection history” was achieved
by establishment of a primary infection by inoculation of birds with 50,000 sporulated E.
acervulina oocysts at day 6 of age (“primed”); the other birds did not receive a primary
infection (“naïve”). The actual transmission experiment started at day 24 of age: one bird
(I) was inoculated with 50,000 sporulated oocysts and was housed together with a non-
inoculated contact bird (C). Oocyst excretion and parameters describing transmission, i.e.
the number of infected C birds and time passed before start of excretion of C birds, were
determinedfromday28today50forsixpairsoffourdifferentcombinationsofIandCbirds
(I–C):naïve–naïve,naïve–primed,primed–naïveandprimed–primed.Immuneparameters,
CD4+, CD8+, ??TCR+and ??TCR+T cells and macrophages in duodenum, were determined
in an additional 25 non-primed, non-inoculated control birds, and in the naïve–naïve and
naïve–primed groups, each group consisting of 25 pairs. Although the numbers of CD4+
T cells and ??TCR+T cells increased after primary infection, none of the immunological
cell types provided an indication of differences in infectivity, susceptibility or transmission
between birds. Oocyst output was significantly reduced in primed I and C birds. Transmis-
sionwasreducedmostintheprimed–primedgroup,butnonethelesstransmissionoccurred
in all groups. This study also showed that acquired immunity significantly reduced oocyst
output after inoculation and contact-infection, but not sufficiently to prevent transmission
to contact-exposed birds.
© 2010 Elsevier B.V. All rights reserved.
∗Corresponding author at: Department of Farm Animal Health, Fac-
ulty of Veterinary Medicine, Utrecht University, P.O. Box 80151, 3508 TD
Utrecht, The Netherlands. Tel.: +31 30 2534447; fax: +31 30 2521887.
E-mail address: f.c.velkers@uu.nl (F.C. Velkers).
1
These two authors contributed equally to this work.
1. Introduction
One of the most prevalent infections in commercial
poultry is coccidiosis, caused by various species of the
genus Eimeria. Infection with Eimeria spp. may cause
enteritis, mortality, welfare problems and economic losses
due to production losses and costs for treatment or
0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2010.07.005

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F.C. Velkers et al. / Veterinary Parasitology 173 (2010) 184–192
185
prevention (Shirley et al., 2005). The majority of the cur-
rent control strategies rely on the use of anticoccidials,
supplemented with live attenuated vaccines. Increasing
resistance among Eimeria species (Chapman, 1997; Peek
and Landman, 2003), legislative restrictions against in-
feed medication, and the lack of success in producing
cost-effective vaccines, stimulate the demand for alter-
native approaches of the problem. More research, aimed
at increasing insight in processes that result in a protec-
tive immune response and towards providing adequate
indicators of protection against infection, might con-
tribute to the development of alternative intervention
strategies.
Immune responses, developed after (repeated) infec-
tion with parasites such as Eimeria spp. affect the
susceptibility and infectivity of individual birds (Rose,
1987; Lillehoj, 1988; Stiff and Bafundo, 1993; Williams,
1995; Claerebout and Vercruysse, 2000). As a result of dif-
ferences in the exposure of birds to infectious oocysts from
the environment, birds in a group are likely to differ in
their infection history, which will result in a heteroge-
neouspopulationwithrespecttoinfectivityandimmunity.
This heterogeneity will cause variability between birds
with respect to oocyst excretion and their response after
subsequent ingestion of oocysts in the population, which
implies that host–pathogen interactions in individuals
will affect the transmission of the parasite between indi-
viduals in a population and vice versa (Severins et al.,
2007).
Most studies carried out in the last decades focused
either on immunity (Lillehoj, 1988; Stiff and Bafundo,
1993; Williams, 1995) or epidemiology (Graat et al., 1998;
Williams, 1998), but it is the mutual interaction between
host and pathogen (“within-host dynamics”), and between
infectious and susceptible hosts in a flock and the envi-
ronment (“between-host dynamics”) that determines the
dynamics of the infection with the pathogen in a flock.
Although this seems straightforward, the outcome of these
interactions may give rise to non-linear effects, which
may result in a counterintuitive and an unpredictable
course of the infection (Roberts and Heesterbeek, 1995,
1998; Graat et al., 1996; Klinkenberg and Heesterbeek,
2007). Therefore, to provide more insight in the course
of the disease coccidiosis in a flock, the dynamics of
the immune reaction in individual hosts and transmis-
sion of the infection between hosts should be studied
together.
The aim of the current study was to examine the
relation between immune responses and transmission
between birds. A transmission experiment was carried
out with broilers with different (artificially induced) infec-
tion histories to Eimeria acervulina, presumed to result in
differentlevelsofacquiredimmunity.Oocystoutputkinet-
ics, intestinal immune responses in individual birds and
transmission characteristics were quantified to determine
whetherimmuneparameterscorrelatedwithinfectivityor
susceptibility of birds, which would be useful to character-
ize differences between individual hosts, and to monitor
and predict transmission of infection between birds. The
approach described in this paper might also be applied to
other (parasitic or protozoan) diseases.
2. Materials and methods
2.1. Chickens and management
At day 0 of age (day 0 of the experiment), 173 male
SPFbroilerchickenswereobtainedfromtheAnimalHealth
Service (AHS, Deventer, The Netherlands). The chicks orig-
inated from a specified pathogen free (SPF) parent flock, a
crossbred of Cobb, Hybro and Ross, kept in an Eimeria spp.
free environment. Until day 25 of age birds were housed
in groups in battery cages (Tecniplast®, Tecnilab-BMI, The
Netherlands). From day 25 onwards, chicks were housed
in pairs on wood shavings in randomly allocated floor pens
(height×width×depth=80cm×60cm×50cm). Control
birds were housed in groups of five birds in battery cages
on wood shavings.
Room temperature was 32◦C at day 0 and was gradu-
allydecreasedto18◦Catday50.Thechicksweresubjected
to a lighting scheme of 23h of light per day. A broiler
ration (12MJ/kg metabolizable energy) without anticoc-
cidial drugs, and drinking water were available ad libitum.
The birds were observed twice daily for signs of ill-
ness or welfare impairment. Birds were housed, handled
and treated following approval by the Animal Experiments
Committee of Utrecht University (Utrecht, The Nether-
lands), in accordance with the Dutch Experiments on
Animals Act.
2.2. Experimental design
An experiment was carried out with pairs of birds con-
sistingofoneinoculated(I)andonenon-inoculatedcontact
(C)bird,referredtoasapair-wisetransmissionexperiment
(Velthuis et al., 2007).
The transmission experiment started when the birds
were 24 days old with inoculation of one bird per pair
(I). Seventy-four I birds were orally inoculated at day 24
with 50,000 sporulated E. acervulina oocysts (Weybridge
W119, supplied by AHS; originally provided by the Cen-
tralVeterinaryLaboratoryinWeybridge,UnitedKingdom),
suspended in 1ml of tap water. The 74 contact birds (C)
were not inoculated. I and C birds were housed pair-
wise in a floor pen from day 25 onwards until the end
of the experiment. Transmission of the infection was sub-
sequently measured by determining for each pair if and
when the contact bird became infected, based on oocyst
excretion.
At the start of the transmission trial, birds differed
in infection history, resulting in different immune status.
The infection history was induced by artificial inocula-
tion of 49 birds at day 6, with a single oral dose of
50,000 sporulated E. acervulina oocysts. At that age, these
birds received a primary infection, denoted by “P” and
referred to as “primed”. The other birds did not receive
such a primary infection (denoted by “N” and referred
to as “non-primed” or “naïve”). From day 6 to day 24 of
age, birds treated equally were housed together in battery
cages.
Thetransmissionexperimentwascarriedoutwithpairs
of birds in four different combinations:

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Table 1
Experimental design: composition and notation of groups, inoculation and measurements.
GroupBirdInoculation day 6
(primary infection)a
Status at day 24a
Inoculation day 24b
# of chicksc
Parameters testedc
Control
1: NN
Control
INN
None
None
Naïve
Naïve
Non-inoculated
Inoculated
25
6
25
Immune parameters
Transmission/oocyst output
Immune parameters
Transmission/oocyst output
Immune parameters
Transmission/oocyst output
Immune parameters
Transmission/oocyst output
Immune parameters
Transmission/oocyst output
Transmission/oocyst output
Transmission/oocyst output
Transmission/oocyst output
1: NNCNN
NoneNaïve Non-inoculated6
25
2: NPINP
None Naïve Inoculated6
25
2: NPCNP
InoculatedPrimed Non-inoculated6
25
3: PN
3: PN
4: PP
4: PP
IPN
CPN
IPP
CPP
Inoculated
None
Inoculated
Inoculated
Primed
Naïve
Primed
Primed
Inoculated
Non-inoculated
Inoculated
Non-inoculated
6
6
6
6
aBefore start of the transmission experiment at day 24, the birds were either primed/had received a primary infection (i.e. were inoculated at day 6 with
50,000 sporulated E. acervulina oocysts) to artificially induce infection history, or were naïve/non-primed (i.e. were not inoculated at day 6).
bAt day 24 birds were either inoculated (I birds) or not (C birds and negative control birds) to start the infectious process in the pair-wise transmission
experiment.
cOocyst output and transmission were determined for I and C birds for six pairs of Groups 1–4 from days 28–50. Immune parameters were determined
in 25 control birds and an additional 25 pairs of I en C birds from Groups 1 (NN) and 2 (NP) at days 28, 31, 33, 36 and 39. Five birds of each group were used
per day of testing.
•Group 1, “NN”, Naïve inoculated bird and naïve contact
bird (INNand CNNbird).
•Group 2, “NP”, Naïve inoculated bird and primed contact
bird (INPand CNPbird).
•Group 3, “PN”, Primed inoculated bird and naïve contact
bird (IPNand CPNbird).
•Group4,“PP”,Primedinoculatedandprimedcontactbird
(IPPand CPPbird).
Notethatforthegroupnotationtheabbreviationforthe
infection status of the inoculated bird, “N” or “P”, precedes
that of the contact bird. For individual birds, the notation
starts with the status in the transmission experiment, i.e.
either inoculated, “I” bird or non-inoculated contact, “C”
bird, followed by a subscript to denote to which group they
belong, i.e. NN, NP, PN or PP.
Transmission and oocyst excretion were quantified for
allfourgroups,consistingofsixpairseach.Immuneparam-
eters were quantified, based on a study by Cornelissen et
al. (2009), for Groups 1 and 2 (NN and NP, respectively),
using 25 additional pairs each; five birds were sacrificed
forquantificationofimmuneparametersateachofthedays
28, 31, 33, 36 and 39. For this immunological study we also
included another five groups of five non-inoculated, non-
primed chickens that served as “negative controls”. Group
composition and treatments are explained in Table 1 and
illustrated with a flow chart in Fig. 1.
2.3. Immune parameters
Five pairs of the NN and NP group and five birds of
the control group were killed by cervical dislocation at
days 28, 31, 33, 36 and 39 (Table 1). One cm of the
duodenal loop was snap-frozen in liquid nitrogen and
stored at −70◦C until further examination by immunohis-
tochemistry. Immunohistological staining by an indirect
immunoperoxidasemethodwasperformedonfrozenduo-
denum sections (8?m thick), collected from chickens
of 28, 31, 33, 36 and 39 days of age as described by
Cornelissen et al. (2009). In short, endogenous perox-
idase was inhibited and were subsequently incubated
for 1h with monoclonal antibodies against CD4+T cells
(1:200; CT-4, Southern Biotech), CD8+T cells (1:200; CT-
8, Southern Biotech), ??TCR+T cells (1:50; TCR2, Southern
Biotech), ??TCR+T cells (1:400; TCR1, Southern Biotech) or
monocytes/macrophages(1:50;KUL-01,SouthernBiotech)
followed by peroxidase-conjugated rabbit anti-mouse Ig
(1:80; P0161, Dakopatts, Denmark). Peroxidase activity
was detected by 0.05% 3,3-diaminobenzidine (DAB) in
0.1M Tris–HCl solution (pH 7.5) containing 0.03% H2O2.
The images were analyzed with Image-Pro Plus (version
5.1, media cybernetics) to quantify the number of stained
T cells per 0.5mm2. Per bird five images were acquired and
analyzed and the mean of the five log10-transformed T cell
counts per bird was used for the statistical analysis.
Immunological data was analyzed per group to deter-
mine whether elevated or decreased levels of the different
cell types could be identified as indicators of infection or
acquired/protective immunity. Therefore, a linear mixed
model was applied using SAS for Windows 9.1 (SAS Insti-
tute Inc., Cary, NC, USA) with log10-transformed mean cell
counts as dependent variable and litter cage as random
factor. Bird type (five different bird types, i.e. INN, CNN,
INP, CNPand control birds), day (days 28, 31, 33, 36 and
39) and the interaction group*day were entered into the
model as explanatory variables. The two-tailed partial F-
test (type III) was used as the elimination criterion for
the model building and the fit of the model was assessed
by the Akaike’s Information Criterion. The best model for
CD4+and CD8+T cells consisted of day and the inter-
action between group*day. The final model for ??TCR+
and ??TCR+cells contained day, group and group*day as
explanatory variables. Differences in T cell counts were
assessed per day between I birds of both groups and con-
trol birds, between C birds of both groups and control
birds, between INNand INPbirds and between CNNand CNP
birds. Bonferroni corrected pair-wise multiple t-tests were
carried out to test the difference between each above men-

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187
Fig.1. Flowchartofthestudydesign,illustratingthetreatmentsatdifferenttimesduringtheexperiment.Atday6,birdseitherreceivedaprimaryinfection
(P) or not (N) to induce different infection histories and were inoculated (I) or not (C) at day 24. From day 25 onwards, four different combinations of I and
C birds were housed pair-wise in floor pens. Immune parameters (left side of the flow chart) were determined for two groups and transmission parameters
(right) for four different groups. The notations for the different bird types in the experiment (INN, CNN, INP, CNP, IPN, CPN, IPP, CPPand control birds) are given
with the number of birds between brackets.
tioned pair of means. Model assumptions were evaluated
by examining normality and equality of variances of the
residuals. The level of statistical significance was set at
P<0.05.
2.4. Droppings collection, processing and oocyst counts
The number of oocysts per gram of faeces (OPG) was
quantified using data from six pairs of birds from Groups 1
to 4. Single individual droppings were collected daily from
days 10 to 23 from primed birds and daily from days 28 to
50 from all I and C birds, or until no oocysts were detected
for three consecutive days. The single droppings were col-
lected and analyzed according to a procedure described by
Velkers et al. (2010). Briefly, each chick was placed in a
cardboardboxwithacleanpapersheetfor1–2h.Thesingle
droppingwasweighedandOPGwasdetermined,according
to a modification of a McMaster oocyst counting chamber
technique described by Long and Rowell (1958).
The detection limit of the McMaster technique was 83
oocystspergoffaecesforafaecalsampleof4g.Ifnooocysts
were found, a modification of the sedimentation–flotation
(SF) technique was applied (Long et al., 1976), which is
a more sensitive technique, though only qualitative (Mes,
2003).
For the oocyst excretion patterns, the average oocyst
production was determined by calculating the mean of the
log10-transformed OPG per day for I and C birds per group.
As a measure for the total number of excreted oocysts, the
areaunderthecurve(AUC)ofoocystoutputwascalculated
for each bird from the daily non-transformed OPG results.
AUCwaslog10-transformed,toobtainnormallydistributed
data,andthemeanlog10-transformedAUC(log10AUC)was
calculated for I and C birds per group. Also the period in
which OPG was intermittently above zero (last day of pos-
itive OPG minus first day of positive OPG results=PERIOD)
and the highest measured log10OPG peak output (PEAK)
were determined.
These data were analyzed per group to determine how
infection history influences oocyst excretion characteris-
tics. Therefore, a linear mixed model was applied with
log10AUC,√PERIOD or PEAK as dependent variable, bird
type (eight different bird types, i.e. I and C birds of each
of the four different groups) as fixed factor and litter cage
as random factor using SAS for Windows 9.1 (SAS Institute
Inc., Cary, NC, USA). The two-tailed partial F-test (type III)
was used as the elimination criterion for the model build-
ing and the fit of the model was assessed by the Akaike’s
InformationCriterion.Bonferronicorrectedpair-wisemul-
tiple t-tests were carried out to test the difference between
each pair of means. Model assumptions were evaluated
by examining normality and equality of variances of the
residuals. The level of statistical significance was set at
P<0.05.

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F.C. Velkers et al. / Veterinary Parasitology 173 (2010) 184–192
Fig. 2. The number of CD4+, CD8+and ??TCR+cells in duodenum sections
from inoculated and contact chickens of the NN group and the NP group
compared to a baseline formed by chickens of the control group at 28, 31,
33, 36 and 39 days of age. CD4+, CD8+and ??TCR+T cells were stained
and the amount of positive cells in an intestinal segment was counted.
Mean of five chickens is shown. Baseline of the graphs is not set at zero,
but at the mean amount of the control values. * Significant differences in
2.5. Transmission between birds
Transmission of the parasite was quantified for Groups
1–4 using the following measures:
•total number of infected contact birds per group;
•infection delay time per pair of birds and the total infec-
tion delay time per group.
The infection delay time, calculated per pair of birds,
is defined as the number of days between the first day at
which contact bird could have started excreting oocysts
and the actual day oocysts were detected for the first time.
The first day at which contact birds could have started
shedding oocysts was two prepatent periods after inocu-
lation (day 32), assuming sporulation time of oocysts to be
negligible (Graat et al., 1994). The prepatent period for E.
acervulina is approximately four days (Edgar, 1955; Joyner
and Long, 1974; McDougald, 2003).
The day the contact bird actually started to excrete
oocysts was based either solely on the McMaster
oocyst counts (McM), or on combined McMaster and
sedimentation–flotationtest
sedimentation–flotation technique was more sensitive
but did not allow quantification of output. When output
started earlier according to the sedimentation–flotation
test, this day was considered first day of excretion. Infec-
tion delay times were added for all pairs per group to
obtain the total infection delay time.
results(McM/SF). The
3. Results
3.1. Immunohistochemistry
Numbers of T cells in birds at days 28, 31, 33, 36 or 39
are shown in Fig. 2. T cell counts did not differ significantly
between non-primed CNNand primed CNPbirds. CNNbirds
had significantly higher CD4+T cell counts compared to
the control birds on day 33. Non-significant increases of
CD4+T cells were observed in I birds on days 31, 33, and
39 (INN) and day 33 (INP), and in CNNbirds on day 39. CNP
birds showed no marked increase in their intestinal CD4+
T cell amount compared to control birds. The number of
??TCR+T cells in INNand INPbirds showed a steep increase
at day 31, and remained high until day 39 (INN) and 36
(INP). The number of ??TCR+T cells of CNNbirds increased
at day 36, whereas the primed CNPbirds did not show a
marked increase. Significant differences in CD8+T cells and
macrophages were not observed between inoculated and
contact birds of the NN and NP groups when compared to
controlbirds.Thenumberof??TCR+Tcellsincontrolbirds
showed high fluctuations for all observed days (data not
shown).
the numbers of the different T cell types between birds of the experimen-
tal group and control birds (P<0.05). Note the difference in the scales.
Plain columns represent measurements before transmission of the para-
site to the contact bird could have taken place, striped columns represent
data when transmission between inoculated and contact birds could have
takenplaceandcheckedcolumnsrepresentdatawhenbothbirdsareable
to infect each other.

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189
Table 2
Oocyst output characteristics for I and C birds for all groups.
GroupBird# of excreting birds
(McM)a
# of excreting birds
(McM/SF)a
OPG positive period
Mean # of days (S.D.)b
Total oocyst output
Mean log10AUC (S.D.)c
Peak oocyst output
Mean log10OPG (S.D.)d
1: NN
1: NN
2: NP
2: NP
3: PN
3: PN
4: PP
4: PP
INN
CNN
INP
CNP
IPN
CPN
IPP
CPP
6
6
6
5
4
6
1
1
6
6
6
6
4
6
5
1
14.67 (3.93)a
13.83 (0.98)a
13.00 (3.10)a
6.00 (5.76)b
2.17 (3.43)b
14.50 (2.17)a
1.00be
1.00be
6.29 (0.30)a
6.83 (0.51)a
6.36 (0.39)a
2.46 (1.42)b
1.59 (1.40)b
5.52 (0.47)a
0.49 (1.20)c
0.24 (0.58)c
5.82 (0.26)a
6.57 (0.53)a
6.03 (0.51)a
2.18 (1.21)b
1.63 (1.44)bc
5.23 (0.44)a
0.48 (1.18)c
0.23 (0.57)c
The mean of the OPG positive period, total oocyst output and peak oocyst output, followed by the standard deviation (S.D.) between brackets, is given per
bird type for each group.
Values within the same column and with different letters (a–c) differ significantly (P<0.05).
aNumber of birds excreting oocysts based solely on McMaster oocyst counts (McM) or on combined McMaster and sedimentation–flotation test results
(McM/SF).
bMean of the OPG positive period (in number of days) of (intermittent) oocyst excretion, which is the last day with OPG larger than zero minus the first
day with OPG larger than zero.
cMean of the log10-transformed AUC of the OPG, which represents total oocyst output during the entire oocyst excretion period.
dMean of the highest log10-transformed OPG, which represents the peak of oocyst output.
eOnly one IPPand one CPPbird were positive, therefore the standard deviation could not be calculated.
3.2. Oocyst excretion patterns and transmission of
infection
After priming, the mean total oocyst excretion was
log10AUC=6.44 (S.D. 0.26). The excretion data after inoc-
ulation or contact-infection are summarized in Table 2.
Supplementary Tables 4–7 show oocyst counts and the
associated infection delay times; in Table 3 the total infec-
tion delay time and the total number of infected contact
birds are given per group.
All non-primed I and C birds (INN, INP, CNN, CPN) excreted
detectablenumbersofoocysts(Table2andSupplementary
Tables 4–6). Oocyst output in the primed IPNbirds was
detected in only four birds, after day 42 of the experi-
ment(SupplementaryTable6),whereasallnon-primedCPN
birdsexcretedoocysts.SixprimedCNPbirdsshowedoocyst
output (five birds positive according to McMaster counts
and six according to sedimentation–flotation results)
(Supplementary Table 5). Five primed IPP birds were
observed to excrete oocysts (one according to McMaster
counts and five birds according to sedimentation–flotation
results), whereas the bird that did not show oocyst excre-
tion according to both tests was the only one with its pen
mate positive (Supplementary Table 7).
Non-primed I and C birds shed oocysts significantly
longer, and had a higher log10AUC and peak output com-
paredtoprimedIandCbirds(Table2).Theoocystexcretion
parameters were significantly higher for CPNbirds com-
pared to CNPbirds and were lowest for CPPbirds. IPNbirds
had a significantly longer OPG positive period and higher
log10AUC than IPPbirds (Table 2).
Oocyst output characteristics, e.g. period of excretion,
total and peak output, were comparable between non-
primedIandCbirds,irrespectiveofgroup(Table2).Primed
IPNand CNPbirds showed comparable oocysts output char-
acteristics and also primed IPP and CPP birds were not
significantly different from each other regarding output
data (Table 2).
In Fig. 3 mean oocyst excretion patterns, based on
McMaster oocyst counts, are visualized for the four groups.
Start of oocyst output in CNPbirds was not delayed (day 33,
Fig. 3B) compared to the CNNbirds (Fig. 3A) but remained
low for the duration of the experiment. On days 33–36
the CNPbirds excreted a lower number of oocysts than the
INPbirds, whereas these birds have experienced the same
environment and infection history in numbers of oocysts
(50,000 in a single dose at inoculation on day 24 for INP
birds and the same dose at priming on day 6 for CNPbirds).
Oocysts were detected in faeces of CNNand CPNbirds from
day 33 onwards (Supplementary Tables 4 and 6). The peak
of the latter was approximately six days later. In the PP
group oocyst output was hardly detectable.
The infection delay times in the NP group were highly
variable between pairs and were reduced when based
on sedimentation–flotation results compared to McMaster
counts (Supplementary Table 5). The lowest total infection
delaytimewasfoundinthenon-primedNNgroup(10days)
and the highest total infection delay time and lowest num-
Table 3
Summary of transmission characteristics: total infection delay time and total number of infected contact birds per group.
GroupTotal infection delay timea
Total number infected C birds
McMMcM/SF McMMcM/SF
1: NN
2: NP
3: PN
4: PP
10
35
17
92
10
10
17
92
6
5
6
1
6
6
6
1
aSum of the infection delay times (based on Supplementary Tables 4–7) for all contact birds together per group.

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F.C. Velkers et al. / Veterinary Parasitology 173 (2010) 184–192
Fig. 3. Mean oocyst output for inoculated and contact birds (n=6), based
on McMaster oocyst counts. (A) Group 1 (NN), (B) Group 2 (NP), (C) Group
3 (PN), (D) Group 4 (PP).
berofinfectedcontactbirds(oneinfectedcontactbird)was
found in the primed PP group (Table 3).
4. Discussion
We studied the effect of heterogeneity in E. acervulina
infection history of broilers on the acquired immunity in
individual birds, and on the transmission characteristics of
the parasite in the population.
4.1. Effects of infection history on immune parameters
None of the immunological cell types showed a signif-
icant sustained elevation level after infection in contact
birds, and no single cell type could be linked to “protection
level” or could predict oocyst output, infectivity, suscepti-
bility or transmission between birds. In non-primed CNN
birds, however, a significant increase in the number of
CD4+Tcellswasobservedaftercontact-infection,butnotin
primed CNPchickens, despite exposure to similar amounts
of oocysts. This suggests that CD4+T cells do not take
part in a protective immune reaction to infection of E.
acervulina in primed chickens, but instead rise in number
duringaprimaryinfectioninvolvingmanyparasites,afind-
ing that is consistent with the study of Trout and Lillehoj
(1996).
CD8+T cells are known to be up-regulated after E.
acervulina infections (Bessay et al., 1996; Swinkels et al.,
2006), and are assumed to play a role in the recovery phase
ofinfections(Lillehoj,1988;Swinkelsetal.,2007).Thebirds
in our experiment, however, did not show a clear change
in CD8+T cell kinetics. This might be due to the gradual
acquisition of parasites after contact-exposure, indicating
that infections established via inoculation with a high dose
may differ from “natural” contact-infection after exposure.
The amount of oocysts ingested might have been too low
to recruit a significant amount of CD8+T cells.
In all chickens, except for the CNP birds an increase
in ??TCR+T cell response to an E. acervulina infection
was observed from day 31 (INN and INP birds) and day
36 (CNN birds) onwards, i.e. at the end of the endoge-
nous phase of the life cycle in the intestinal tract (Fig. 2).
Because ??TCR+T cells are associated with development
of intestinal epithelia (Boismenu and Havran, 1994) and
may recognize damaged tissue (Schild et al., 1994), the
increase is probably a reaction to intestinal damage after
E. acervulina infections. In inoculated birds we see that the
??TCR+T cell response remains high when the response
in the non-primed contact chickens develops as well. This
may indicate that the response to infection due to inoc-
ulation, the first infection cycle, is persistently high, but
an alternative explanation may be that re-infection with
excreted oocysts from the environment, a second infec-
tioncycle,isresponsible.Thelatterexplanationisverywell
possible,becauseoocystexcretionremainshighduringthe
second infection cycle, indicating considerable tissue dam-
ageandhencerecruitmentof??TCR+Tcells(Fig.2and3A).
PrimedCNPbirdsdidnotshowanincreaseof??TCR+Tcells.
Itisnotclearwhetherthissuggeststhataprimaryresponse
to infection results in different ??TCR+T cell kinetics com-
paredtoaprotectiveresponseafterre-infectionorthatthis
finding can be solely explained by the reduced oocyst out-
put and hence limited tissue damage in primed CNPbirds.
Although we might have identified T cell markers for
either a current E. acervulina infection (CD4+T cells) or
intestinal damage due to an E. acervulina infection (??TCR+
T cells), none of the single cell types showed a sustained
elevatedlevelafterinfectioninprimedornon-primedcon-
tact broilers. Therefore it can be concluded that none of the
studied cell types correlates well with “protection level”
nor was able to predict oocyst output, infectivity, suscepti-

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F.C. Velkers et al. / Veterinary Parasitology 173 (2010) 184–192
191
bility of previously infected birds or transmission between
birds.
4.2. Effects of primary infection on oocyst output and
transmission between birds
It is known that after ingestion of oocysts, the induced
immune response can reduce oocyst output after re-
infection(Lillehoj,1988;Williams,1995;Blakeetal.,2005).
In this experiment, the infection history reduced oocyst
output in inoculated birds and contact-exposed birds. The
non-primed CPNbirds showed a delayed peak of excre-
tion compared to the non-primed CNNbirds, suggesting
a delayed transmission of Eimeria. Although oocyst out-
put by the primed IPNbirds was hardly detectable, these
birds were apparently sufficiently infectious to establish
contact-infection, indicating the usefulness of transmis-
sion experiments, which mimics natural infections better
than experiments with “artificially” inoculated birds only.
Oocyst output by the primed CNPbirds was significantly
lower compared to that of the non-primed CNNand CPN
birds, indicating a reduced susceptibility. However, trans-
mission of infection did occur, which suggests that the
acquired immunity was not sufficient to resist the high
number of oocysts excreted by the INPbird. Whether or not
this was the case for the PP combination cannot be deter-
mined, as it cannot be determined whether the apparent
absence of oocysts in the faeces of CPPbirds is due to the
factthatthebirdswerenotinfectedorthatthenumberwas
below the detection limit of the tests. Limitations of detec-
tionaredemonstratedbythetwoCPNbirdsandoneCPPbird
that excreted oocysts, whereas their inoculated pen mates
did not show oocyst output for the entire duration of the
experiment. This suggests that small numbers of oocysts
remain undetectable for both techniques. A quantitative
PCR technique might be more appropriate to improve the
detection limit for faecal samples with small numbers of
oocysts (Blake et al., 2008; Morgan et al., 2009; Velkers et
al., 2010).
Non-primed inoculated and contact birds do not dif-
fer in infection history before day 24 (inoculation), but
do differ in timing and dose of ingestion of oocysts after
that. Inoculated and contact birds most likely repeatedly
ingested variable numbers of oocysts from the environ-
ment after day 28, but inoculated birds also received
a single inoculation dose of 50,000 oocysts at day 24.
Becausedurationofoutputandtotaloutputwerecompara-
ble between non-primed inoculated and contact birds, the
inoculation was apparently irrelevant. However, a single
inoculation 18 days earlier (priming) did change the excre-
tion pattern: priming itself caused a single high peak, and
after day 24 total output and duration were significantly
lower and shorter than in non-primed birds. Apparently,
time intervals between oocyst ingestion are sometimes
crucial for subsequent oocyst output (primed birds), and
in some cases not at all (non-primed I and C birds). Only
transmission experiments or experiments in which birds
are allowed to re-infect themselves automatically provide
a realistic timing of ingestions. It shows that simple chal-
lenge experiments, using high doses of oocysts, may give
different results, and that oocyst output cannot be simply
translated to predictions on outcome of infection or spread
of the disease.
Another observation on timing of oocyst ingestions can
be made by comparing the second infection cycles of non-
primed inoculated birds (INNand INP), and of primed CNP
birds. These infection cycles correspond with the excretion
around days 32–36 and the ??TCR+T cell responses at days
36 and 39. Before the second infection cycle all these birds
had experienced the same infection history with respect to
numbers of oocysts (50,000 in a single dose), but they dif-
fered in the moment in life these oocysts had been given.
Because the inoculated birds showed much higher oocyst
excretionandelevated??TCR+Tcelllevelsthantheprimed
contact birds, this suggests that inoculation at day 24 of INN
and INPbirds resulted in a less effective protective immune
responsethanthepriminginoculationatday6ofCNPbirds.
Apparently,thereisadelayintheimmuneresponse,which
may have considerable impact on dynamics in a larger
population, and which is not yet covered by present mod-
els described by Klinkenberg and Heesterbeek (2007) and
Severins et al. (2007).
In conclusion, contrary to our expectation that the
measured immune parameters could indicate acquired
immunity by predicting level of protection against oocyst
output and transmission, no cell types were identified in
this study to confirm this assumption. However, it was
shown that primary infection could significantly reduce
oocyst output after secondary infection. Furthermore, the
natural infection of contact birds by exposure to inocu-
latedbirdshasshownthatundetectablylowoocystoutputs
can cause contact-infections, where the level of the sub-
sequent oocyst output in contact birds depends on the
level of acquired immunity obtained through the infection
history of these birds. The combined study of within-
host and between-host dynamics also has revealed that a
delaybetweeninfectionanddevelopmentofimmunitycan
occur, which can have great impact on infection dynamics
in flocks.
Acknowledgement
We would like to thank Hans Vernooij for his valuable
help and advice during the statistical analysis.
Appendix A. Supplementary data
Supplementary
can
doi:10.1016/j.vetpar.2010.07.005.
data associated
inthe
withthisarti-
cle befound, onlineversion, at
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