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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 Kaldhusdal
a a b a

, 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 Available 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 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 identication by morphometric and polymerase chain reaction techniques
Anita Haug1,2*, Anne-Gerd Gjevre1, Per Thebo2, Jens G. Mattsson2 and Magne Kaldhusdal1
1 2

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.

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 (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 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 04350 in Study 1, and 79 )50 03950 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 0

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Eimeria infections in commercial broilers

below detection limit of 100 OPG; 1 0100 to 999 OPG; 201000 to 9999 OPG; 3 010 000 to 49 999 OPG; 4 0more than 50 000 OPG) using an ordinal logistic model. The models were assessed for fit using standard procedures.

163

Results 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.

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-

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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

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.

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) 20 Litter 27.1 (23) 26 Litter 32.9 (28)

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

2000/01 (Study 1) (n085) 32a Faeces 42.4 (36) 32a Faeces 76.0 (79) 2003/04 (Study 2) (n0104)

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 Percentage of Eimeria spp. in positive flocksg E. acervulina E. mitis E. necatrix E. tenella E. praecox E. maxima E. brunetti
a

71.4 92.9 57.1

86.1 94.4 55.6

95.8 97.2 23.9 n 061 100.00 Not detected ( B1.6) 1.64h 77.05 9.84 24.59 Not detected ( B1.6)

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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 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
a

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

Test statistics for each of the models tested

Coefficient P value 95% confidence interval

Odds ratio

5.45 1.82 5.62 10.21 (0.30 0.50 0.60 1.10 2.89 2.01 3.91 7.84

0.000 0.145 0.002 0.000 0.367 0.250 0.108 0.031 0.000 0.050 0.000 0.000

2.78 0.81 1.89 2.96 0.98 0.57 0.13 0.10 1.65 1.00 1.84 3.13

to to to to to to to to to to to to

10.65 4.06 16.68 4.06 4.82 1.36 1.33 2.10 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 0below detection limit of 100 OPG; 1 0100 to 999 OPG; 201000 to 9999 OPG; 3 010 000 to 49 999 OPG; 40 above 50 000 OPG.
b

Eimeria infections in commercial broilers

Infection levels (log10 OPG) a) 6 7 b) c)

165

2000-01

2003-04

2000-01

2003-04

2000-01

2003-04

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 ocks, show the minimum, 25%, median, 75% and maximum infection levels for each region and time period.

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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 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.

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

a) 80 60 40 20 Percent flocks

b)

c)

d) 80 60 40 20

e)

f)

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.

166 A. Haug et al.

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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.

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,

The distribution of Eimeria species combinations in Norwegian broiler ocks in 2003/04, based on faecal samples collected at slaughtera
Species combinations E. E. E. E. E. E. E. acervulina acervulina'E. acervulina'E. acervulina'E. acervulina'E. acervulina'E. acervulina'E. Number of flocks (n061) Percentage of flocks tested 10 33 4 8 3 2 1 16 54 7 13 5 3 2

Number of species 1 2 3 4 5
a

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

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

Mean age 32.1 days.

Eimeria infections in commercial broilers

167

Table 4. Description of the discrepancies between a morphometric tentative identication where the oocysts were divided into three length categories and PCR identication
PCR resultsa Oocyst length categoriesb AM'NTP Number of samples with discrepancies 5 2 Discrepancy between methods Hypothesis

AC

AC

AM'NTP' BM

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 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 No oocysts in BM, maximum length 21.3 mm

Oocyst lengths within natural length range of AC c AC length range wider than reported

Oocyst lengths within natural length range of AC and/or AC length range wider than reported AC length range wider than reported

AC'MA

AM'NTP

Oocyst lengths within natural length ranges of AC and MA

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Oocyst lengths within natural length range of AC and percentage of MA in mixed infection low

AC'TE

AM'NTP' BM

Oocyst lengths within natural length range of TE

AC'TE

AM

Oocyst lengths within natural length range of TE and/ or percentage of TE in mixed infection low

AC'TE 'MA

AM'NTP

3 3

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

AC'TE'PR

AM'NTP' BM

Oocyst lengths within natural length ranges of TE and PR

AC'TE'PR

NTP'BM

Oocyst lengths within natural length range of AC and or percentage AC in mixed infection low

AC'TE'PR' MA

AM'NTP

Percentage of MA in mixed infection low

AC 0E. acervulina; TE 0E. tenella; MA 0E. maxima; PR0E. praecox; NE0E. necatrix. AM 0small oocysts (518.8 mm) (i.e. tentative Eimeria species are E. acervulina or E. mitis); NTP 0medium-sized oocysts (18.8 to 23.8 mm) (i.e. tentative Eimeria species are E. necatrix, E. tenella or E. praecox); BM 0large 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

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

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Eimeria infections in commercial broilers

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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 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.

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

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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

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