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Avian Pathology
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Coccidial infections in commercial broilers:

epidemiological aspects and comparison of Eimeria
species identification by morphometric and
polymerase chain reaction techniques
Anita Haug

a b

Kaldhusdal

, Anne-Gerd Gjevre , Per Thebo , Jens G. Mattsson & Magne

Department of Pathology , National Veterinary Institute , Ullevlsveien 68, Pb. 8156,

N-0033, Oslo, Norway
b

Department of Parasitology (SWEPAR) , National Veterinary Institute and Swedish

University of Agricultural Sciences , SE-751 89, Uppsala, Sweden
Published online: 08 Apr 2008.

To cite this article: Anita Haug , Anne-Gerd Gjevre , Per Thebo , Jens G. Mattsson & Magne Kaldhusdal
(2008) Coccidial infections in commercial broilers: epidemiological aspects and comparison of Eimeria species
identification by morphometric and polymerase chain reaction techniques, Avian Pathology, 37:2, 161-170, DOI:
10.1080/03079450801915130
To link to this article: http://dx.doi.org/10.1080/03079450801915130

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Avian Pathology (April 2008) 37(2), 161170

Coccidial infections in commercial broilers: epidemiological

aspects and comparison of Eimeria species identification by
morphometric and polymerase chain reaction techniques
Anita Haug1,2*, Anne-Gerd Gjevre1, Per Thebo2, Jens G. Mattsson2 and
Magne Kaldhusdal1
1

Department of Pathology, National Veterinary Institute, Ullevalsveien 68, Pb. 8156, N-0033 Oslo, Norway, and
Department of Parasitology (SWEPAR), National Veterinary Institute and Swedish University of Agricultural Sciences,
SE-751 89 Uppsala, Sweden

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The objective of this study was to add to existing knowledge of the epidemiology and the aetiology of
coccidial infections in commercial broiler flocks. Polymerase chain reaction (PCR) and morphometric
identification of the Eimeria species were compared as means of differentiation in the field samples of faeces
and litter. For morphometry, the Eimeria species were categorized into three groups based on lengths of the
oocysts. Two random samples of commercial broilers were studied, one during 2000/01 and the other during
2003/04. The prophylactic regime (in-feed narasin), husbandry and methods applied were broadly the same
for both subpopulations. Coccidial infection prevalence increased from approximately 45% to approximately
75% during this period, but infection levels (oocysts per gram of faeces) did not significantly change. There
were substantial geographical differences in both prevalence and infection levels. A change in Eimeria species
profile occurred during the study period. Five Eimeria species were identified at slaughter, by PCR targeting
the ITS-1 region of the genome; Eimeria acervulina (100%), Eimeria tenella (77%), Eimeria maxima
(25%), Eimeria praecox (10%) and Eimeria necatrix (2%). PCR and morphometric tentative identification
were in complete agreement in only 49% of the cases.

Introduction
Despite the advances in poultry husbandry, nutrition
and chemotherapy that have made clinical outbreaks of
coccidiosis rather infrequent, subclinical coccidiosis
continues to be one of the poultry industrys most
common and expensive diseases worldwide (McDougald, 2003). The broiler industry in particular relies on
continuous in-feed prophylaxis with application of anticoccidial drugs. Much due to the industrys and the
publics awareness of the emergence of drug resistance
and possible drug residues, the EU Commission has
proposed a phasing out of such use by 31 December
2012 (EU Commission, 2003). This forthcoming ban is
dependent on the industry establishing alternative control measures for rearing broilers, without compromising
commercial production performance, animal welfare and
health. The application of specific diagnostics, as well as
studying the epidemiology and intensity of the infections, is important for carrying out rational and effective
control measures (McDougald, 2003).
Species differentiation within the coccidia has traditionally been based on comparing several parasite
characteristics and host responses (Long et al., 1976;
Long & Reid, 1982). This diagnostic procedure is not
only expensive and time-consuming, but can also be
unreliable since the different species have overlapping
properties and the intra-species variation is substantial

(Joyner & Long, 1974; Pellerdy, 1974; Long & Joyner,

1984; Thebo et al., 1998). Knowledge of Eimeria species
at the genomic level is continuously emerging, and
objective molecular methods for Eimeria species differentiation have been developed (Stucki et al., 1993; Tsuji
et al., 1997; Schnitzler et al., 1998, 1999; Gasser et al.,
2001, 2005; Su et al., 2003; Lien et al., 2007; Haug et al.,
2007). Nevertheless, the practical implementation of
these techniques in routine diagnostics and epidemiological studies of chicken coccidiosis have so far been
limited (Lew et al., 2003; Gasser et al., 2005; Blake et al.,
2006; Morris et al., 2007a,b).
Substantial work on coccidiosis based on experimental
infections and drug and vaccine trials has been presented
over many years. However, reports on infection prevalence, infection levels and frequencies of the different
Eimeria species in commercial broiler flocks are few and
sporadic. Often the reports are not comparable due to
the differences in management and production systems,
sample materials, sampling periods, sampling methods
and prophylactic measures applied. More knowledge of
the aetiology and population dynamics of mixed coccidial infections in commercial broilers is therefore needed.
The main objective of this work was to expand the
knowledge of the epidemiology of coccidial infections in
commercial broiler flocks by studying the geographical

*To whom correspondence should be addressed. Tel:
47 23216424. Fax:
47 23216303. E-mail: anita.haug@vetinst.no
ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/08/20161-10 # 2008 Houghton Trust Ltd
DOI: 10.1080/03079450801915130

162 A. Haug et al.

distribution of coccidial infections, prevalence, infection

levels, and the Eimeria species present in commercial
broiler populations. We also wanted to compare PCRbased identification of Eimeria spp. with a tentative
identification based on measurement of oocyst lengths.
The results of two independent field studies, conducted
in the Norwegian broiler population during the years
2000 to 2004, are presented.

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Materials and methods

Norwegian broiler production. The Norwegian coastal climate is
temperate, whereas the inland climate is harsher, with subarctic
conditions in the north. There are approximately 550 broiler farms in
Norway. The broiler industry is concentrated in three regions (Figure 1).
Of the total number of broiler farms, approximately 23%, 55% and 22%
are in Regions 1, 2 and 3, respectively. The chickens are reared on wood
shavings on concrete floors, in insulated, free-range broiler houses. The
material of the construction of the house varies between wood, metal
and concrete. The indoor climate is regulated by in-floor heating
systems (some use electric heaters or hot-air systems), and a mechanical
ventilation system (overpressure and underpressure systems are equally
widespread) controlled by a climate computer. Automatic cup feeders
and nipple drinkers are standard. According to Norwegian legislation,
the maximum bird density is 34 kg live weight/m2, corresponding to
about 23 birds/m2 on the day of slaughter. During this study, the
average flock size was approximately 12 000 birds (ranging from
approximately 3000 to approximately 40 000 birds), and the slaughter
age was approximately 31 days of age. Occasionally, some flocks are
slaughtered at a higher age to produce larger broilers. The most
common commercial hybrids reared during the study period were Ross
208 and Cobb 500. The Norwegian commercial feed companies produce
pellets with oat, wheat and soy flour as main ingredients. The pellets are
sometimes combined with whole grains. In-feed anticoccidial drugs are
used prophylactically until 5 days prior to slaughter. The drug used in
these studies was almost exclusively narasin (a polyether ionophore); the
very few flocks medicated with other drugs also received an ionophore.
Anticoccidial vaccines are not used in Norwegian broilers, and
antibacterial growth promoters have not been used in Norway since
1995. Between successive grow-outs, used litter is removed and the
broiler house is cleaned and chemically disinfected.
Study population and sample collection. Two observational field studies
were conducted. In study 1, litter and faecal samples were collected
from 85 commercial broiler farms (one flock per house and farm)
between April 2000 and December 2001. The flocks were selected by
stratified random sampling from the total Norwegian broiler population. Fifty-four per cent of the samples were collected in the autumn/
winter season (1 October to 31 March). Of the 85 farms selected, 18
(21%), 49 (58%) and 18 (21%) were in Regions 1, 2 and 3, respectively.
Samples were collected at approximately days 20 and 26 (litter) of age
and on the day of slaughter (faeces). Median age at slaughter was 32
days. Broilers slaughtered when younger than 37 days were defined as
standard broilers, and broilers slaughtered after 36 days were defined as
large broilers. Two flocks were classified as large broilers with a
maximum slaughter age of 39 days.
Study 2 was conducted between December 2003 and November 2004,
with 13% of the samples collected in the autumn/winter season. Samples
of faeces were collected on the day of slaughter from 98 standard broiler
farms (one flock per house and farm) throughout Norway, selected by
simple random sampling. Six more farms were included specifically
because of their late slaughter time (large broilers). Maximum slaughter
age was 69 days, and the median age at slaughter age was 31 days (n
104). Of the 104 farms, 20 (19%), 70 (67%) and 14 (13%) were in
Regions 1, 2 and 3, respectively.
The litter samples in Study 1 were collected by regional poultry
advisers. One sample of 100 ml surface litter was collected from each of
five evenly distributed areas of the selected house on each farm. Each
sample was put into a zipped plastic bag, kept cool in an expanded
polyester box using a cooler brick, and sent by express mail to the
laboratory where the five litter samples were immediately mixed and

pooled into one sample. A 10 g subsample was weighed and mixed

thoroughly with 40 ml of 2% potassium dichromate, and stored at 48C
for a maximum of approximately 11 months until further processing.
The faecal samples in Studies 1 and 2 were collected from the
transport containers of each study flock at the slaughter house. Faeces
were collected from 10 different locations of the transport containers
and pooled into a zipped plastic bag. In Study 1 the faecal samples were
sent and treated like the litter samples. Maximum storage times
approximately 11 months; however, about 80 per cent of the samples
were processed within 3 months. In Study 2 the samples were by express
mail and processed immediately on receipt at the laboratory or within 3
days (stored at 48C).
Determination of infection levels and classification of oocysts. The levels
of oocysts per gram of sample (OPG) were determined using a standard
McMaster technique as previously described by the Ministry of
Agriculture, Fisheries and Food (1986). In Study 1, each stored sample
(containing 10 g faeces or litter) was transferred to a plastic beaker, and
water was added until the sample weighed approximately 140 g (1:15
dilution). In Study 2, a 3 g faecal sample was transferred to a beaker
before adding 42 ml water (1:15 dilution). In Study 1, all oocysts under
the grid in one McMaster chamber were counted (0.15 ml); alternatively, three columns (0.075 ml) of two chambers were counted and the
sum of the two counts was recorded. In Study 2, the mean of the counts
of oocysts under the grid of two chambers was calculated. The
minimum detection levels in Study 1 and 2 corresponded to 100 OPG
and 50 OPG, respectively.
A modified saturated salt flotation technique (Ministry of Agriculture, Fisheries and Food, 1986) was used to isolate oocysts for length
measurements. Using a calibrated ocular micrometer at 400x magnification (Long & Reid, 1982), 50 random oocysts from each sample were
measured and categorized into three groups: an AM group (small
oocysts, 518.8 mm; tentatively Eimeria acervulina and/or Eimeria
mitis), an NTP group (medium-sized oocysts, 18.9 to 23.8 mm;
tentatively Eimeria necatrix, Eimeria tenella and/or Eimeria praecox)
or a BM group (large oocysts, ]23.9 mm; tentatively Eimeria brunetti
and/or Eimeria maxima). The total number of oocysts measured was
(23
28
36) 50 4350 in Study 1, and 79 50 3950 in Study 2.
Identification of Eimeria species by PCR. The oocysts were concentrated
and isolated from the faeces by a flotation technique using saturated
sodium chloride solution, and were then washed free from the salt by
repeated centrifugation and resuspension in tap water (Shirley, 1995).
To be able to identify any Eimeria species present in only very small
numbers in mixed infections, only samples containing approximately
100 000 oocysts per 50 ml test sample were selected for testing. A total of
61 faecal samples from Study 2 were tested; namely, 15% flocks from
Region 1, 67% from Region 2, and 18% from Region 3. The infection
levels in the selected samples ranged from 500 OPG to 1 485 000 OPG.
Ten per cent of the samples were collected from flocks of large broilers.
The DNA preparation and PCR were performed as previously described
by Haug et al. (2007); oocysts were ruptured by pestle grinding, DNA
extracted using modified Gene-Releaser protocol and Eimeria species
identified by PCR using species-specific primers targeting ITS-1. Only
one-half of the volumes of sample and reagents compared with the
original protocol were used. Based on morphometry, an assumption of
E. acervulina being ubiquitous was made. Hence, E. acervulina functioned as an internal control of the PCRs. The theoretical minimal
detection level is found to be 0.4 to 2 oocysts for each Eimeria species
per PCR (Haug et al. 2007).
Statistical analysis. Statistical analysis of data was performed using the
statistical package Stata/SE 9.2 (Stata Corp, College Station, Texas,
USA). Initial descriptive analyses included establishing of regional
prevalences, as well as descriptive tabular and graphical examination of
the data. Further exploration of data was performed using regression
analysis. In all of the analyses the study variables were study (Studies 1
and 2) and region (Regions 1, 2 and 3). If appropriate, the interaction
between these two was also tested in all models.
Three different models were established based upon a binomial
outcome (OPG 0) using a logistic model, a continuous outcome (log10
OPG) using a median regression model and an ordinal outcome (0 

Eimeria infections in commercial broilers

below detection limit of 100 OPG; 1 100 to 999 OPG; 21000 to 9999
OPG; 3 10 000 to 49 999 OPG; 4 more than 50 000 OPG) using an
ordinal logistic model. The models were assessed for fit using standard
procedures.

Results

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Prevalence of infection. There was significantly higher

infection prevalence at slaughter in 2003/04 compared
with 2000/01 (Figure 1 and Tables 1 and 2). The
infection prevalence was lowest in Region 1 and highest
in Region 3 in both studies. The difference was
statistically significant when comparing Region 1 with
Region 3, and Region 2 with Region 3. However, the
increase in prevalence during the study period was higher
in Region 1 than the other two regions (Figure 1 and
Table 2). In two of the flocks in Study 1, oocysts were
found in both litter samples, but not in the faecal sample
collected at slaughter.
Level of infection at slaughter. The observed decrease in
infection levels from 2000/01 to 2003/04 could not be
statistically confirmed (Tables 1 and 2). However, there
were similar geographical differences in both studies,
with the lowest infection levels found in Region 1 and the
highest in Region 3. The differences were only found
statistically significant when comparing Regions 3 and 1
(Figure 2 and Table 2). During the study period, the
median infection levels seemed to decrease in Region 2
and even more so in Region 3, but remained almost
constant in Region 1 (Figure 2). Nevertheless, the

163

infection level range seemed to increase in Region 1.

The data did not allow statistical confirmation of these
observations. There were, however, statistically significant differences in the distribution of infection level
categories, both between study periods and between all
three regions (Figure 3 and Table 2). No cases of clinical
coccidiosis were reported in any of the flocks selected for
Studies 1 and 2.
Large broilers compared with the total study population.
In Studies 1 and 2 there were two and six flocks of large
broilers (slaughter age ]37 days), respectively. Two
flocks were slaughtered at 39 days of age, one flock at
41 days, one flock at 46 days, two flocks at 49 days, and
one flock at 69 days of age. The last flock was registered
with the slaughter age of approximately 45 days. All the
flocks of large broilers in Studies 1 and 2 were coccidia
positive. The median infection levels of large broilers
from both surveys were B14 000 OPG (range 350 to 30
750).
Tentative species categories and Eimeria species identified
at slaughter. The distribution of oocysts among the
different length categories at slaughter varied between
the two study periods and the three regions (Figure 4
and Table 1). Flocks positive for the NTP group were
found most frequently at slaughter in both studies;
however, the frequency of NTP-positive and AM-positive flocks were almost the same in Study 2. The
frequencies BM-positive flocks at slaughter varied con-

Figure 1. Geographical distribution of commercial broiler farms in Norway, as well as the geographical prevalence of coccidial infection
during 2000/01 and 2003/04.

164 A. Haug et al.

Table 1.

Parameters of coccidial infection in Norwegian broilers during 2000/01and 2003/04

2000/01 (Study 1)
(n85)

Study
Approximate age (days) of
chickens at sampling
Sample source
Prevalence of infection (%)
(number of positive flocks)
Median infection levels
(OPG)b (infection level range)

2003/04 (Study 2)
(n104)

20

26

32a

32a

Litter
27.1 (23)

Litter
32.9 (28)

Faeces
42.4 (36)

Faeces
76.0 (79)

18 400 (400 to 540 000) 38 600 (500 to 1 080 000) 37 800 (400 to 12 000 000) 13 750 (100 to 1 485 000)

Percentage of oocyst categories positive flocksc

AM groupd
78.3
NTP groupe
95.7
BM groupf
69.6

71.4
92.9
57.1

86.1
94.4
55.6

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Percentage of Eimeria spp. in positive flocksg

E. acervulina
E. mitis
E. necatrix
E. tenella
E. praecox
E. maxima
E. brunetti

95.8
97.2
23.9
n 61
100.00
Not detected ( B1.6)
1.64h
77.05
9.84
24.59
Not detected ( B1.6)

Faeces collected on day of slaughter. Median slaughter age is 32 days and 31days in Studies 1 and 2, respectively.
Median infection levels in coccidia-positive flocks.
c
Percentage of coccidia-positive flocks with presence of oocysts within each group size.
d
Small oocysts; that is, tentative Eimeria species are E. acervulina or E. mitis.
e
Medium-sized oocysts; that is, tentative Eimeria species are E. necatrix, E. tenella or E. praecox.
f
Large oocysts; that is, tentative Eimeria species are E. brunetti or E. maxima.
g
Eimeria species, given as percentage of investigated coccidia-positive flocks in 2003/04.
h
Found in one flock, at 69 days of age.
b

siderably between Studies 1 and 2, but were clearly lower

than the frequencies of flocks positive for the two other
categories in both studies (Table 1). Region 1 showed the
largest shift in oocyst length group composition with
time, from 100% to 20% BM-positive flocks and from
45% to 100% AM-positive flocks, in 2000/01 and 2003/
04, respectively. The other two regions showed a slight
increase of AM-positive and NTP-positive flocks and a
decrease of BM-positive flocks, Region 3 showing the
largest changes. When considering both litter and faecal
Table 2.
Outcome variable
Infection prevalencea
Study 2 versus study 1
Region 2 versus region 1
Region 3 versus region 2
Region 3 versus region 1

Test statistics for each of the models tested

Odds ratio

Coefficient

5.45
1.82
5.62
10.21

Infection levelsa,b
Study 2 versus study 1
Region 2 versus region 1
Region 3 versus region 2
Region 3 versus region 1
Categorized infection levelsa,c
Study 2 versus study 1
Region 2 versus region 1
Region 3 versus region 2
Region 3 versus region 1

samples collected in Study 1, there was also a tendency

towards an increase in the occurrence of small oocysts
and a decrease in occurrence of large oocysts during
rearing (Table 1).
The differences between regions in mean relative
frequencies of each species category were considerable
in 2000/01, but insignificant in 2003/04. However, there
was a considerable change in mean relative species
composition with time in all three regions (Figure 4).
In large broilers, the two flocks in the 2000/01 study

0.30
0.50
0.60
1.10
2.89
2.01
3.91
7.84

P value

95% confidence interval

0.000
0.145
0.002
0.000

2.78
0.81
1.89
2.96

to
to
to
to

10.65
4.06
16.68
4.06

0.367
0.250
0.108
0.031

0.98
0.57
0.13
0.10

to
to
to
to

4.82
1.36
1.33
2.10

0.000
0.050
0.000
0.000

1.65
1.00
1.84
3.13

to
to
to
to

5.06
4.01
8.29
19.62

Oocysts per gram of faeces at slaughter.

Continuous OPG levels in coccidia-positive flocks.
c
Infection levels: 0 below detection limit of 100 OPG; 1 100 to 999 OPG; 21000 to 9999 OPG; 3 10 000 to 49 999 OPG; 4
above 50 000 OPG.
b

b)

c)

a)

165

Infection levels (log10 OPG)

Eimeria infections in commercial broilers

2003-04

2000-01

2000-01

2003-04

2000-01

2003-04

possessed oocysts where the majority belonged to the

NTP group. In 2003/04, the coccidial infections in the
large broilers were generally dominated by AM oocysts
or had a maximum of 45% of NTP oocysts.
Five of the seven Eimeria species known to infect
chickens were detected by PCR in the faecal samples
from 2003/04 (Table 1). E. acervulina was found in all the
flocks tested, but neither E. brunetti nor E. mitis was
detected. Two or more species were found in 84% of the
samples. Monospecific infections with E. acervulina, and
mixed infections with E. acervulina and E. tenella were
most common (Table 3). All five species were detected in
Region 2, but only E. acervulina, E. tenella and E.
maxima were detected in Regions 1 and 3.
E. necatrix was found in a single flock that was
slaughtered at 69 days of age. The infection level of this
flock was moderate (9200 OPG), but the faecal sample
contained all five species hereby confirmed in Norway.

a)

All of the other five samples from flocks of large broilers,

the oldest being 49 days of age, contained E. acervulina
in combination with E. tenella.

Relationship between oocyst length and Eimeria species.

Comparison of PCR results with the tentative identification based on oocyst length measurements (i.e. belonging to category AM, NTP or BM) showed complete
agreement only in 49% (30 of 61) of the flocks in Study
2. Agreement was defined as followed: when an Eimeria
species was identified by PCR, oocysts of corresponding
oocyst length category was also detected by morphometry, and vice versa*when an oocyst of one length
category was identified by morphometry, at least one of
the Eimeria species belonging to that category was
identified by PCR. When considering actual oocyst
lengths measured and the differences in detection levels

b)

c)

e)

f)

80
60
40
20
Percent flocks

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Figure 2. Infection levels (log10 OPG) at slaughter in (2a) Region 1, (2b) Region 2 and (2c) Region 3 during 2000/01 and 2003/04.
The diagrams, based on data from coccidia-positive flocks, show the minimum, 25%, median, 75% and maximum infection levels for each
region and time period.

d)
80
60
40
20

0
1
2
3
4
Infection levels at slaughter age

Figure 3. Infection level categories 0 to 4: (3a) Region 1 in 2000/01, (3b) Region 2 in 2000/01, (3c) Region 3 in 2000/01, (3d) Region 1
in 2003/04, (3e) Region 2 in 2003/04 and (3f) Region 3 in 2003/04.

Downloaded by [187.122.224.170] at 07:44 13 March 2015

166 A. Haug et al.

Figure 4.

Regional mean relative frequencies of oocysts of each length category during (4a) 2000/01 and (4b) 2003/24.

between the two methods, plausible causes were found in

27 of the 31 discrepancies (Table 4).

Discussion
The frequency of coccidial infections in Norwegian
broiler chickens was studied during two different time
periods under very similar conditions. Faecal samples
were collected at slaughter from birds receiving narasin
as an anticoccidial feed additive, and were examined by a
modified McMaster technique. During this time-frame
there was an increase in infection prevalence of approximately 30%. Coccidia in commercial broilers are often
assumed to be ubiquitous (Stayer et al., 1995; McDougald, 2003). Yet, in reports on infection prevalence in
broilers worldwide, the prevalences vary from less than
10% to more than 90% (Oikawa et al., 1979; Braunius,
1986b; McDougald et al., 1986, 1997; Williams et al.,
1996; Graat et al., 1998; Al Natour et al., 2002).
However, the methods applied, time of sampling, animal
husbandry and meat production management differ
Table 3.

The distribution of Eimeria species combinations in Norwegian broiler flocks in 2003/04, based on faecal samples collected at
slaughtera

Number of species
1
2
3
4
5
a

substantially between these studies. We found prevalence

to vary considerably with time even under similar
conditions. The low infection prevalence observed in
Region 1 in 2000/01 suggests that coccidial infections in
broilers might not always be as extensive as often
assumed.
Whereas the prevalence of infection increased, there
seemed to be a tendency of decreasing infection levels
during the study period (not statistically significant). The
infection levels varied substantially from hundreds to
millions of oocysts per gram of faeces, without clinical
coccidiosis being reported. Similar OPG levels were
found in broilers in France also without observing
clinical coccidiosis (Williams et al., 1996).
The geographical variation in infection prevalence and
the infection levels at slaughter was substantial and both
increased with latitude. The magnitude of decrease in
infection level during the study period also seemed to
correspond with latitude. Regional differences in prevalence have previously been described in other countries
(Oikawa et al., 1979; Braunius, 1988). In our study,

Species combinations
E.
E.
E.
E.
E.
E.
E.

Mean age 32.1 days.

acervulina
acervulina
E.
acervulina
E.
acervulina
E.
acervulina
E.
acervulina
E.
acervulina
E.

tenella
maxima
tenella
E.
tenella
E.
tenella
E.
tenella
E.

maxima
praecox
maxima
E. praecox
maxima
E. praecox
E. necatrix

Number of flocks (n61) Percentage of flocks tested

10
33
4
8
3
2
1

16
54
7
13
5
3
2

Eimeria infections in commercial broilers

167

Table 4. Description of the discrepancies between a morphometric tentative identification where the oocysts were divided into three
length categories and PCR identification
PCR resultsa

AC

Oocyst length
categoriesb
AM
NTP

Number of samples with

discrepancies

Discrepancy between methods

Hypothesis

No Eimeria species of NTP group

detected by PCR
A few oocysts in NTP, maximum
length 22.5 mm
Two oocysts in NTP, maximum
length 23.8 mm
No Eimeria species of NTP or BM
groups detected by PCR

Oocyst lengths within natural length

range of AC c
AC length range wider than reported

AC

AM
NTP

BM

AC
MA

AM
NTP

Downloaded by [187.122.224.170] at 07:44 13 March 2015

AC
TE

AM
NTP

BM

AC
TE

AM

AC
TE
MA

AM
NTP

3
3

AC
TE
PR

AM
NTP

BM

AC
TE
PR

NTP
BM

AC
TE
PR

MA

AM
NTP

A few oocysts in NTP and one in

BM (26.3 mm)
50% of oocysts spread within
NTP, two in lower BM range
No Eimeria species of NTP group
detected by PCR and no oocysts of
BM group detected by morphometry
approximately 50% spread within
NTP
One or few oocysts in NTP, maximum length 20 mm
No Eimeria species of BM group
detected by PCR
One or few oocysts in BM, maximum length 26.3 mm
No oocysts of NTP group detected
by morphometry
No oocysts in NTP, maximum
length 20 mm
No oocysts of BM group detected
by morphometry
No oocysts in BM, maximum length
20 mm
No oocysts in BM, most oocyst in
NTP in the lower half range
No Eimeria species of BM group
detected by PCR
No oocysts in BM, maximum length
25 mm
No Eimeria species of BM group
detected by PCR and no oocysts of
AM group detected by morphometry
No oocysts in AM, minimum length
20mm
No oocysts of BM group detected
by morphometry

Oocyst lengths within natural length

range of AC and/or AC length range
wider than reported
AC length range wider than reported

Oocyst lengths within natural length

ranges of AC and MA

Oocyst lengths within natural length

range of AC and percentage of MA
in mixed infection low

Oocyst lengths within natural length

range of TE

Oocyst lengths within natural length

range of TE and/ or percentage of TE
in mixed infection low

Percentage of MA in mixed infection

low
Percentage of MA in mixed infection
low and/or oocyst lengths within
natural length range of MA

Oocyst lengths within natural length

ranges of TE and PR

Oocyst lengths within natural length

range of AC and or percentage AC in
mixed infection low

Percentage of MA in mixed infection

low

No oocysts in BM, maximum length

21.3 mm
a

AC E. acervulina; TE E. tenella; MA E. maxima; PRE. praecox; NEE. necatrix.
AM small oocysts (518.8 mm) (i.e. tentative Eimeria species are E. acervulina or E. mitis); NTP medium-sized oocysts (18.8 to
23.8 mm) (i.e. tentative Eimeria species are E. necatrix, E. tenella or E. praecox); BM large oocysts ( ]23.9 mm) (i.e. tentative Eimeria
species are E. brunetti or E. maxima).
c
Oocyst length range as reviewed by Pellerdy (1974).
b

approximately one-half of the samples were collected in

autumn/winter in the 2000/01 survey, in contrast to just
over 10% in 2003/04. Both Braunius (1986a) and Graat
et al. (1998) found coccidial infections to occur more
often in autumn and winter in The Netherlands.

Assuming a possible seasonal effect on the occurrence

of coccidial infections, the true difference in prevalence
between the two time periods studied might be even
greater. The Norwegian broilers are reared under highly
controlled conditions. However, maintaining a stable

Downloaded by [187.122.224.170] at 07:44 13 March 2015

168 A. Haug et al.

and optimal environment in the broiler chicken house

can be challenging in a coastal climate or at very cold
temperatures. We were not able to find any apparent
trends in mean temperatures, precipitation or relative
humidity for the three regions. Therefore, it seems less
probable that latitude and climate differences are key
factors for the regional and seasonal differences observed.
The use of one single anticoccidial compound
throughout the broilers life, and throughout the study
period, makes emerging drug tolerance an important
factor to consider. Narasin has been used as almost the
sole anticoccidial compound in Norwegian broilers since
1996, and ionophores have dominated since 1988
(Norwegian Food Safety Authority, www.mattilsynet.no). Ionophore resistance develops slowly due to
complexity in mode of action (Braunius, 1986a; Jeffers,
1989; Chapman, 1997). However, a decrease in efficacy
(i.e. development of tolerance) can develop gradually
(Braunius, 1986a; McDougald et al., 1986). Braunius
(1986a) observed a rapid decline in prevalence of
coccidial infection after introduction of a polyether
ionophore (monensin), but prevalence increased to premonensin levels within a few years. Cross resistance
between polyether ionophores is well documented (Weppelman et al., 1977; McDougald et al., 1986; Voeten,
1989). Nonetheless, caution must be exercised when
using oocyst counts as a means of evaluating anticoccidial efficacy (Reid, 1975) as factors such as initial
infection level, Eimeria species involved, reproduction
potential, crowding effect and acquired immunity influence the oocyst output (Brackett & Bliznick, 1952;
Williams, 1973; Henken et al., 1994; Graat et al., 1996;
Williams, 2001).
Possible differences in meat production management
might also account for our findings. Broiler meat
production in Norway increased from about 43 000
tonnes to 54 000 tonnes during the study period. This led
to increased flock sizes, rather than more farms. It is
possible that larger flocks are associated with increased
prevalence of coccidial infections due to having more
animals producing large amounts of oocysts in a very
confined space. Region 1 had the smallest flock sizes,
and Region 3 the largest in 2000/01. These differences in
flock sizes had decreased in 2003/04. Region 1 is also a
more recently developed broiler farm district. This might
influence rearing conditions and management, but also
the expansion potential of the infection, which again
could be the reason for this being the region with the
largest changes. This region has a generally better
commercial performance than the other two regions.
Apparently low infection prevalences can result from
infection levels being below the detection limit of the
method used. Owing to disintegration of oocysts with
time, there is a possibility that the storage of samples
before processing in Study 1 might have had an impact
on the detection of flocks with infection levels just
around detection limits. Hence, the prevalence and
infection levels detected in Study 1 could have been
higher than reported here. However, we were not able to
find any apparent relation between storage time and
degree of deformity of the oocysts or the OPG level, nor
did we find differences in distribution of storage time
between infected and uninfected flocks (unpublished
observation), making this an unlikely explanation for the
substantial discrepancies between the two surveys.

Also, in Study 1 we used stratified random sampling

of flocks, and in Study 2 simple random selection. This
led to Region 2 being over-represented and Region 3
being under-represented in Study 2. This should be kept
in mind when extrapolating the results to the total
broiler population.
When comparing the prevalences of each oocyst size
category during rearing in 2000/01, there was a minor
increase of small oocysts and a decrease of large oocysts
with age. A similar shift in oocyst composition was
observed by Stayer et al. (1995) in litter samples. The
change in species composition during rearing could be
attributable to the high reproductive potential of E.
acervulina (Brackett & Bliznick, 1952; Williams, 2001)
and its ability to suppress other Eimeria species in mixed
infections (Williams, 1973).
We found differences in species composition both with
time and between regions. Fluctuations in species
composition of coccidial infection and their intensities
are well documented (Long, 1964; Hodgson et al., 1969;
Braunius, 1986b; Hamet, 1986). They might be due to
fluctuations in immunity (Williams, 1995) or differences
in specific efficacy of the ionophores (Ryley & Wilson,
1975; Jeffers, 1989; Schildknecht & Untawale, 1989).
Braunius (1986a) found that extended or repeated use of
an anticoccidial compound tended to change the spectrum of activity.
No recordings on the presence of Eimeria species in
chickens have previously been conducted in Norway. All
seven Eimeria species have been confirmed to be present
in Swedish poultry (Thebo et al., 1998); however, this
was not in broiler chickens only. We were here able to
identify five species (i.e. E. acervulina, E. tenella, E.
maxima, E. praecox and E. necatrix) in the Norwegian
broilers at slaughter in 2003/04. More than 80% of
infections in our study were mixed. Our findings of
Eimeria species in commercial broilers are for the most
part in agreement with other European reports (Kucera,
1990; Williams et al., 1996; Graat et al., 1998). We
detected E. acervulina on every farm tested, as occurred,
for instance, in France (Williams et al., 1996) and in the
United Kingdom (Williams, 2006). However, the prevalence of E. maxima was rather low in Norwegian
broiler flocks, which might be due to their early
slaughter age. The OPG for E. maxima often peaks at
5 to 8 weeks of age (Voeten & Braunius, 1981; Williams,
1995), although it may appear earlier in a flock if its
sensitivity to the prophylactic drug being used becomes
reduced (Williams, 2006). The apparent absence of E.
brunetti might also be due to the early slaughter age; on
the other hand, this species is often reported to be rare in
broilers (Oikawa et al., 1979; Long & Reid, 1982;
Williams et al., 1996; Graat et al., 1998). We did not
detect E. mitis, and the prevalence of E. praecox was
rather low. These species are believed to be underdiagnosed due to the lack of macroscopic lesions
(McDougald, 2003). However, E. mitis occurs frequently
in European broilers (Kucera, 1990; Williams et al.,
1996; Williams, 2006) and elsewhere (McDougald et al.,
1997; Morris et al., 2007a). There is no obvious
explanation for our different findings. The apparent
regional differences in species occurrence could be due to
small sample sizes in the two regions where only three of
the five species were detected.
Several laboratories have performed tentative Eimeria
species differentiation based on oocyst length categories

Downloaded by [187.122.224.170] at 07:44 13 March 2015

Eimeria infections in commercial broilers

(Oikawa et al., 1979; Kucera, 1990; Chapman &

Johnson, 1992; Stayer et al., 1995; Waldenstedt, 1998).
We only found perfect agreement in about one-half of
the cases when size category assessment is compared
with PCR results. Interestingly, the majority of our
discrepancies could be explained by one or a few oocysts
being outside the limits of the neighbouring size
category, but they were within the reported size range
of the species identified by the PCR analysis. The size
ranges are wide and the overlaps between species are
substantial (Pellerdy, 1974; Long & Reid, 1982).
Observed discrepancies could very well be the result of
the higher sensitivity with PCR, with possibility to detect
coccidia down to individual sporozoites in each reaction
(Haug et al., 2007). The samples for PCR used in this
study contained approximately 100 000 oocysts, which
correspond to roughly 4000 oocysts per PCR. Based on
the assumption of a detection level of two oocysts per
PCR, screening such a large sample makes us able to
detect Eimeria species present in a mixture with a relative
frequency down to 0.05% (two oocysts per PCR/4000).
In contrast, in the morphological tests we measured 50
random oocysts. Screening 50 oocysts per PCR, Eimeria
species with a relative frequency of 4% could still be
identified. Infrequent species will always remain undetected using morphometry, and there is also a risk that
the relatively small subsample investigated is not representative of the total sample.
The discrepancies in four of the samples could neither
be attributed to oocysts being within reported natural
length variation, nor differences in detection levels of the
two methods. It is thus tempting to speculate on the
possibility that the oocyst size ranges of Eimeria spp. are
even wider than previously reported (Pellerdy, 1974;
Long & Reid, 1982). It has also been demonstrated that
the oocyst size varies due both to environmental and
physical factors (Jones, 1932; Joyner, 1982). Nevertheless, with PCR being qualitative, using size distribution
of the oocysts with the oocyst length categories as rough
guides can be useful as a rapid tool to identify the
predominating species group in a mixed infection.
An understanding of the aetiology and the epidemiology of subclinical coccidiosis is essential in coccidiosis
control. Even though clinical coccidiosis is rather
sporadic in the modern broiler industry, subclinical
coccidiosis remains one of the most important infections
causing decline in production performance. The significance of the presence of coccidia at different infection
levels, the relative impact of the different Eimeria species
on broiler performance, and further evaluation of the
correlation between flocks classified as being at high risk
and their actual performance will be addressed in a
subsequent study.

Acknowledgements
The authors would like to thank all farmers, veterinarians and the staff at the slaughterhouses for assisting in
the sampling process. They also thank Youssef Rohoma
and Reidun Bolstad for technical assistance in the
laboratory, Eystein Skjerve for statistical guidance, Ole
Einar Tveito for providing meteorological data, and Ray
Williams and Bjrn Gjerde for fruitful discussions and
help with the manuscript. This work has been supported

169

by grants from The Research Council of Norway and the

Norwegian Centre for Poultry Science.

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