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Coccidial infect ions in commercial broilers:epidemiological aspects and comparison of Eimeriaspecies ident if icat ion by morphometr ic andpolymerase chain reaction techniquesAnit a Haug

ab

, Anne-Gerd Gjevrea

, Per Thebob

, Jens G. Mattssonb

& Magne

Kaldhusdala

aDepartment of Pathology, National Veterinary Inst it ute, Ull evlsveien 68, Pb. 8156,

N-0033, Oslo, Norwayb

Depart ment of Parasit ology (SWEPAR), National Veterinary Inst it ute and SwedishUniversit y of Agri cultural Sciences, SE-751 89, Uppsala, Sweden

Available online: 08 Apr 2008

To cite t his art icle: Anita Haug, Anne-Gerd Gj evre, Per Thebo, Jens G. Mattsson & Magne Kaldhusdal (2008): Coccidialinfecti ons in commercial broilers: epidemiological aspects and comparison of Eimeria species ident if icat ion bymorphometric and polymerase chain reaction techniques, Avian Pathology, 37:2, 161-170

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Coccidial infections in commercial broilers: epidemiologicalaspects and comparison ofEimeria species identification bymorphometric and polymerase chain reaction techniques

Anita Haug1,2*, Anne-Gerd Gjevre1, Per Thebo2, Jens G. Mattsson2 andMagne Kaldhusdal1

1Department of Pathology, National Veterinary Institute, Ullevalsveien 68, Pb. 8156, N-0033 Oslo, Norway, and2Department of Parasitology (SWEPAR), National Veterinary Institute and Swedish University of Agricultural Sciences,SE-751 89 Uppsala, Sweden

The objective of this study was to add to existing knowledge of the epidemiology and the aetiology ofcoccidial infections in commercial broiler flocks. Polymerase chain reaction (PCR) and morphometricidentification 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 theoocysts. Two random samples of commercial broilers were studied, one during 2000/01 and the other during2003/04. The prophylactic regime (in-feed narasin), husbandry and methods applied were broadly the samefor both subpopulations. Coccidial infection prevalence increased from approximately 45% to approximately75% during this period, but infection levels (oocysts per gram of faeces) did not significantly change. Therewere substantial geographical differences in both prevalence and infection levels. A change in Eimeria speciesprofile occurred during the study period. Five Eimeria species were identified at slaughter, by PCR targetingthe 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 identificationwere 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 (McDou-

gald, 2003). The broiler industry in particular relies on

continuous in-feed prophylaxis with application of antic-

occidial 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 con-trol 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 infec-

tions, is important for carrying out rational and effective

control measures (McDougald, 2003).Species differentiation within the coccidia has tradi-

tionally 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 ofEimeria species

at the genomic level is continuously emerging, and

objective molecular methods for Eimeria species differ-

entiation 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 epidemiolo-

gical 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 experimentalinfections and drug and vaccine trials has been presented

over many years. However, reports on infection pre-

valence, 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 cocci-

dial 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: [email protected]

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

ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/08/20161-10 # 2008 Houghton Trust Ltd

DOI: 10.1080/03079450801915130

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distribution of coccidial infections, prevalence, infectionlevels, and the Eimeria species present in commercialbroiler populations. We also wanted to compare PCR-based identification of Eimeria spp. with a tentativeidentification based on measurement of oocyst lengths.

The results of two independent field studies, conductedin the Norwegian broiler population during the years2000 to 2004, are presented.

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 feedersand 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, andantibacterial 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 popula-

tion. 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 (n0

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 4 8C

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 expressmail 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); alterna-

tively, 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 Agricul-

ture, Fisheries and Food, 1986) was used to isolate oocysts for length

measurements. Using a calibrated ocular micrometer at 400x magnifi-

cation (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))5004350 in Study 1, and 79)5003950 in Study 2.

Identification ofEimeria 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. Onlyone-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 func-

tioned 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 (OPG0) using a logistic model, a continuous outcome (log 10OPG) using a median regression model and an ordinal outcome (00

162 A. Haug et al.

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below detection limit of 100 OPG; 10100 to 999 OPG; 201000 to 9999

OPG; 3010 000 to 49 999 OPG; 40more than 50 000 OPG) using an

ordinal logistic model. The models were assessed for fit using standard

procedures.

Results

Prevalence of infection. There was significantly higher

infection prevalence at slaughter in 2003/04 comparedwith 2000/01 (Figure 1 and Tables 1 and 2). Theinfection prevalence was lowest in Region 1 and highest

in Region 3 in both studies. The difference wasstatistically significant when comparing Region 1 withRegion 3, and Region 2 with Region 3. However, theincrease in prevalence during the study period was higherin Region 1 than the other two regions (Figure 1 andTable 2). In two of the flocks in Study 1, oocysts werefound in both litter samples, but not in the faecal samplecollected at slaughter.

Level of infection at slaughter. The observed decrease ininfection levels from 2000/01 to 2003/04 could not be

statistically confirmed (Tables 1 and 2). However, therewere similar geographical differences in both studies,with the lowest infection levels found in Region 1 and thehighest in Region 3. The differences were only foundstatistically significant when comparing Regions 3 and 1(Figure 2 and Table 2). During the study period, themedian infection levels seemed to decrease in Region 2

and even more so in Region 3, but remained almostconstant in Region 1 (Figure 2). Nevertheless, the

infection level range seemed to increase in Region 1.The data did not allow statistical confirmation of these

observations. There were, however, statistically signifi-cant differences in the distribution of infection levelcategories, both between study periods and between allthree regions (Figure 3 and Table 2). No cases of clinicalcoccidiosis were reported in any of the flocks selected forStudies 1 and 2.

Large broilers compared with the total study population.

In Studies 1 and 2 there were two and six flocks of largebroilers (slaughter age ]37 days), respectively. Twoflocks were slaughtered at 39 days of age, one flock at41 days, one flock at 46 days, two flocks at 49 days, andone flock at 69 days of age. The last flock was registeredwith the slaughter age of approximately 45 days. All theflocks of large broilers in Studies 1 and 2 were coccidiapositive. The median infection levels of large broilersfrom both surveys were B14 000 OPG (range 350 to 30750).

Tentative species categories and Eimeria species identified

at slaughter. The distribution of oocysts among thedifferent length categories at slaughter varied betweenthe two study periods and the three regions (Figure 4and Table 1). Flocks positive for the NTP group werefound most frequently at slaughter in both studies;however, the frequency of NTP-positive and AM-posi-

tive flocks were almost the same in Study 2. Thefrequencies 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.

Eimeria infections in commercial broilers 163

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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 withtime, 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

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

Table 1. Parameters of coccidial infection in Norwegian broilers during 2000/01and 2003/04

Study

2000/01 (Study 1)

(n085)

2003/04 (Study 2)

(n0104)

Approximate age (days) of

chickens at sampling

20 26 32a 32a

Sample source Litter Litter Faeces Faeces

Prevalence of infection (%)

(number of positive flocks)

27.1 (23) 32.9 (28) 42.4 (36) 76.0 (79)

Median infection levels

(OPG)b (infection level range)

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 71.4 86.1 95.8

NTP groupe 95.7 92.9 94.4 97.2

BM groupf 69.6 57.1 55.6 23.9

Percentage of Eimeria spp. in positive flocksg n061

E. acervulina 100.00

E. mitis Not detected (B1.6)

E. necatrix 1.64h

E. tenella 77.05

E. praecox 9.84

E. maxima 24.59

E. brunetti Not detected (B1.6)

aFaeces collected on day of slaughter. Median slaughter age is 32 days and 31days in Studies 1 and 2, respectively.bMedian infection levels in coccidia-positive flocks.cPercentage of coccidia-positive flocks with presence of oocysts within each group size.dSmall oocysts; that is, tentative Eimeria species are E. acervulina or E. mitis.eMedium-sized oocysts; that is, tentative Eimeria species are E. necatrix, E. tenella or E. praecox.fLarge oocysts; that is, tentative Eimeria species are E. brunetti or E. maxima.gEimeria species, given as percentage of investigated coccidia-positive flocks in 2003/04.hFound in one flock, at 69 days of age.

Table 2. Test statistics for each of the models tested

Outcome variable Odds ratio Coefficient P value 95% confidence interval

Infection prevalencea

Study 2 versus study 1 5.45 0.000 2.78 to 10.65

Region 2 versus region 1 1.82 0.145 0.81 to 4.06Region 3 versus region 2 5.62 0.002 1.89 to 16.68

Region 3 versus region 1 10.21 0.000 2.96 to 4.06

Infection levelsa,b

Study 2 versus study 1 (0.30 0.367 0.98 to 4.82




Categorized infection levelsa,c

Study 2 versus study 1 2.89 0.000 1.65 to 5.06




aOocysts per gram of faeces at slaughter.bContinuous OPG levels in coccidia-positive flocks.cInfection levels: 00below detection limit of 100 OPG; 10100 to 999 OPG; 201000 to 9999 OPG; 3010 000 to 49 999 OPG; 40

above 50 000 OPG.

164 A. Haug et al.

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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 theflocks 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.

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

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

Relationship between oocyst length and Eimeria species.

Comparison of PCR results with the tentative identifica-tion based on oocyst length measurements (i.e. belong-

ing 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 Eimeriaspecies was identified by PCR, oocysts of corresponding

oocyst length category was also detected by morpho-

metry, and vice versa*when an oocyst of one lengthcategory was identified by morphometry, at least one ofthe Eimeria species belonging to that category was

identified by PCR. When considering actual oocystlengths measured and the differences in detection levels

2

3

4

5

6

7

2000-01 2003-04 2000-01 2003-04Infectionlevels(log10

OPG)

2000-01 2003-04

a) b) c)

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 le vels for each

region and time period.

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4

Percentflocks

Infection levels at slaughter age

a) b) c)

d) e) f)

20

40

60

80

80

60

40

20

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.


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between the two methods, plausible causes were found in

27 of the 31 discrepancies (Table 4).

Discussion

The frequency of coccidial infections in Norwegianbroiler chickens was studied during two different timeperiods under very similar conditions. Faecal sampleswere collected at slaughter from birds receiving narasinas an anticoccidial feed additive, and were examined by amodified McMaster technique. During this time-framethere was an increase in infection prevalence of approxi-mately 30%. Coccidia in commercial broilers are oftenassumed to be ubiquitous (Stayer et al., 1995; McDou-gald, 2003). Yet, in reports on infection prevalence in

broilers worldwide, the prevalences vary from less than10% 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, animalhusbandry and meat production management differ

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 pre-valence have previously been described in other countries

(Oikawa et al., 1979; Braunius, 1988). In our study,

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

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 Species combinations Number of flocks (n061) Percentage of flocks tested

1 E. acervulina 10 16

2 E. acervulina'E. tenella 33 54

E. acervulina'E. maxima 4 7

3 E. acervulina'E. tenella'E. maxima 8 13

E. acervulina'E. tenella'E. praecox 3 5

4 E. acerv

ulina'

E. tenella'

E. maxima'

E. praecox 2 35 E. acervulina'E. tenella'E. maxima'E. praecox'E. necatrix 1 2

aMean age 32.1 days.

166 A. Haug et al.

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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

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 Oocyst length

categoriesbNumber of samples with

discrepancies

Discrepancy between methods Hypot hesis

AC AM'NTP 5 No Eimeria species of NTP group

detected by PCR

Oocyst lengths within natural length

range of AC c

2 A few oocysts in NTP, maximum

length 22.5 mm

AC length range wider than reported

Two oocysts in NTP, maximum

length 23.8 mm

AC AM'NTP'

BM

1 No Eimeria species of NTP or BM

groups detected by PCR


range of AC and/or AC length range

wider than reported

1 A few oocysts in NTP and one in

BM (26.3 mm)

AC length range wider than reported

50% of oocysts spread within

NTP, two in lower BM range

AC'MA AM'NTP 2 No Eimeria species of NTP group

detected by PCR and no oocysts of

BM group detected by morphome-

try


ranges of AC and MA

2 approximately 50% spread within

NTP


range of AC and percentage of MAin mixed infection low

One or few oocysts in NTP, max-

imum length 20 mm

AC'TE AM'NTP'

BM

8 No Eimeria species of BM group

detected by PCR


range of TE

One or few oocysts in BM, max-

imum length 26.3 mm

AC'TE AM 1 No oocysts of NTP group detected

by morphometry


range of TE and/ or percentage of TE

in mixed infection low

No oocysts in NTP, maximum

length 20 mm

AC'TE 'MA AM'NTP 3 No oocysts of BM group detected

by morphometry

Percentage of MA in mixed infection

low

3 No oocysts in BM, maximum length20 mm

Percentage of MA in mixed infectionlow and/or oocyst lengths within

natural length range of MA

No oocysts in BM, most oocyst in

NTP in the lower half range

AC'TE'PR AM'NTP'

BM

1 No Eimeria species of BM group

detected by PCR


ranges of TE and PR

No oocysts in BM, maximum length

25 mm

AC'TE'PR NTP'BM 1 No Eimeria species of BM group

detected by PCR and no oocysts of

AM group detected by morphome-

try


range of AC and or percentage AC in

mixed infection low

No oocysts in AM, minimum length

20mm

AC'TE'PR'MA

AM'NTP 1 No oocysts of BM group detectedby morphometry

Percentage of MA in mixed infectionlow

No oocysts in BM, maximum length

21.3 mm

aAC0E. acervulina; TE0E. tenella; MA0E. maxima; PR0E. praecox; NE0E. necatrix.bAM0small oocysts (518.8 mm) (i.e. tentative Eimeria species are E. acervulina or E. mitis); NTP0medium-sized oocysts (18.8 to

23.8 mm) (i.e. tentative Eimeria species are E. necatrix, E. tenella or E. praecox); BM0large oocysts (]23.9 mm) (i.e. tentative Eimeria

species are E. brunetti or E. maxima).cOocyst length range as reviewed by Pellerdy (1974).


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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 relativehumidity for the three regions. Therefore, it seems less

probable that latitude and climate differences are key

factors for the regional and seasonal differences ob-served.

The use of one single anticoccidial compoundthroughout 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 since1996, and ionophores have dominated since 1988

(Norwegian Food Safety Authority, www.mattilsy-

net.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 pre-

monensin levels within a few years. Cross resistance

between polyether ionophores is well documented (Wep-pelman et al., 1977; McDougald et al., 1986; Voeten,1989). Nonetheless, caution must be exercised when

using oocyst counts as a means of evaluating antic-occidial efficacy (Reid, 1975) as factors such as initial

infection level, Eimeria species involved, reproductionpotential, crowding effect and acquired immunity influ-

ence 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 meatproduction in Norway increased from about 43 000

tonnes to 54 000 tonnes during the study period. This ledto increased flock sizes, rather than more farms. It is

possible that larger flocks are associated with increased

prevalence of coccidial infections due to having moreanimals 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 mightinfluence rearing conditions and management, but also

the expansion potential of the infection, which again

could be the reason for this being the region with thelargest 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 withtime, 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 beenhigher 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 timebetween 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 keptin 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 oocystswith age. A similar shift in oocyst composition was

observed by Stayer et al. (1995) in litter samples. Thechange in species composition during rearing could beattributable to the high reproductive potential of E.acervulina (Brackett & Bliznick, 1952; Williams, 2001)and its ability to suppress other Eimeria species in mixedinfections (Williams, 1973).

We found differences in species composition both with

time and between regions. Fluctuations in speciescomposition 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 spec-

trum 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 presentin Swedish poultry (Thebo et al., 1998); however, thiswas not in broiler chickens only. We were here able toidentify five species (i.e. E. acervulina, E. tenella, E.maxima, E. praecox and E. necatrix) in the Norwegianbroilers at slaughter in 2003/04. More than 80% of

infections in our study were mixed. Our findings ofEimeria species in commercial broilers are for the mostpart in agreement with other European reports (Kucera,

1990; Williams et al., 1996; Graat et al., 1998). Wedetected E. acervulina on every farm tested, as occurred,for instance, in France (Williams et al., 1996) and in theUnited Kingdom (Williams, 2006). However, the pre-

valence of E. maxima was rather low in Norwegianbroiler flocks, which might be due to their early

slaughter age. The OPG for E. maxima often peaks at5 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 becomesreduced (Williams, 2006). The apparent absence of E.brunetti might also be due to the early slaughter age; onthe 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 notdetect E. mitis, and the prevalence of E. praecox wasrather low. These species are believed to be under-

diagnosed due to the lack of macroscopic lesions(McDougald, 2003). However, E. mitis occurs frequentlyin European broilers (Kucera, 1990; Williams et al.,1996; Williams, 2006) and elsewhere (McDougald et al.,1997; Morris et al ., 2007a). There is no obviousexplanation for our different findings. The apparentregional differences in species occurrence could be due to

small sample sizes in the two regions where only three ofthe five species were detected.

Several laboratories have performed tentative Eimeriaspecies differentiation based on oocyst length categories

168 A. Haug et al.

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(Oikawa et al ., 1979; Kucera, 1990; Chapman &Johnson, 1992; Stayer et al., 1995; Waldenstedt, 1998).We only found perfect agreement in about one-half ofthe cases when size category assessment is comparedwith PCR results. Interestingly, the majority of our

discrepancies could be explained by one or a few oocystsbeing outside the limits of the neighbouring sizecategory, but they were within the reported size range

of the species identified by the PCR analysis. The sizeranges are wide and the overlaps between species aresubstantial (Pellerdy, 1974; Long & Reid, 1982).

Observed discrepancies could very well be the result ofthe 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 thisstudy contained approximately 100 000 oocysts, whichcorrespond to roughly 4000 oocysts per PCR. Based on

the assumption of a detection level of two oocysts perPCR, screening such a large sample makes us able to

detect Eimeria species present in a mixture with a relativefrequency 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, Eimeriaspecies with a relative frequency of 4% could still beidentified. Infrequent species will always remain unde-tected using morphometry, and there is also a risk thatthe relatively small subsample investigated is not repre-

sentative of the total sample.The discrepancies in four of the samples could neither

be attributed to oocysts being within reported naturallength variation, nor differences in detection levels of the

two methods. It is thus tempting to speculate on thepossibility that the oocyst size ranges of Eimeria spp. areeven wider than previously reported (Pellerdy, 1974;Long & Reid, 1982). It has also been demonstrated that

the oocyst size varies due both to environmental andphysical factors (Jones, 1932; Joyner, 1982). Neverthe-less, with PCR being qualitative, using size distributionof the oocysts with the oocyst length categories as rough

guides can be useful as a rapid tool to identify thepredominating species group in a mixed infection.

An understanding of the aetiology and the epidemiol-ogy of subclinical coccidiosis is essential in coccidiosis

control. Even though clinical coccidiosis is rathersporadic in the modern broiler industry, subclinicalcoccidiosis remains one of the most important infections

causing decline in production performance. The signifi-cance of the presence of coccidia at different infectionlevels, the relative impact of the different Eimeria species

on broiler performance, and further evaluation of thecorrelation between flocks classified as being at high riskand their actual performance will be addressed in asubsequent study.

Acknowledgements

The authors would like to thank all farmers, veterinar-ians and the staff at the slaughterhouses for assisting in

the sampling process. They also thank Youssef Rohomaand Reidun Bolstad for technical assistance in the

laboratory, Eystein Skjerve for statistical guidance, OleEinar Tveito for providing meteorological data, and Ray

Williams and Bjrn Gjerde for fruitful discussions andhelp with the manuscript. This work has been supported

by grants from The Research Council of Norway and theNorwegian Centre for Poultry Science.

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