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Efficacy of early treatment with toltrazuril inprevention of coccidiosis and necrotic enteritis inchickensAlaa Aldin Alnassana, Awad Ali Shehatab, Marianne Kotschb, Wieland Schrödlb, MonikaKrügerb, Arwid Daugschiesa & Berit Bangouraa

a Faculty of Veterinary Medicine, Centre for Infectious Diseases, Institute of Parasitology,University of Leipzig, Leipzig, Germanyb Faculty of Veterinary Medicine, Centre for Infectious Diseases, Institute forBacteriology and Mycology, University of Leipzig, Leipzig, GermanyAccepted author version posted online: 12 Jul 2013.Published online: 13 Aug 2013.

To cite this article: Alaa Aldin Alnassan, Awad Ali Shehata, Marianne Kotsch, Wieland Schrödl, Monika Krüger, ArwidDaugschies & Berit Bangoura , Avian Pathology (2013): Efficacy of early treatment with toltrazuril in prevention ofcoccidiosis and necrotic enteritis in chickens, Avian Pathology, DOI: 10.1080/03079457.2013.823476

To link to this article: http://dx.doi.org/10.1080/03079457.2013.823476

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ORIGINAL ARTICLE

Efficacy of early treatment with toltrazuril in prevention ofcoccidiosis and necrotic enteritis in chickens

Alaa Aldin Alnassan1, Awad Ali Shehata2, Marianne Kotsch2, Wieland Schrodl2,Monika Kruger2, Arwid Daugschies1 and Berit Bangoura1*

1Faculty of Veterinary Medicine, Centre for Infectious Diseases, Institute of Parasitology, University of Leipzig, Leipzig,Germany, and 2Faculty of Veterinary Medicine, Centre for Infectious Diseases, Institute for Bacteriology and Mycology,University of Leipzig, Leipzig, Germany

In the present study, efficacy of the toltrazuril treatment for prevention of coccidiosis and necrotic enteritiswas tested. Ninety-six 14-day-old commercial broiler chickens were caged and divided into eight groups(n�12), designated groups 1 to 8. Chickens of groups 1 to 6 were inoculated orally at 18 days of age with25,000 oocysts of Eimeria tenella and 75,000 oocysts of Eimeria brunetti. At 22 days of age, chickens ofgroups 1 to 6 were infected with 109 colony-forming unit Clostridium perfringens. Chickens of group 1 weretreated with 75 parts/106 toltrazuril in drinking water for 8 h on two consecutive days up to 12 h beforeEimeria infection, while chickens of groups 2 to 5 were treated with the same dose of toltrazuril at 12 h, 36 h,60 h and 84 h after Eimeria infection, respectively. The non-treated group 6 served as a positive control.Chickens in group 7 were treated with toltrazuril at 17 and 18 days of age, and those of group 8 remaineduninfected and non-treated as a negative control. The feed conversion ratio was higher in the positive controlcompared with other groups. The mortality rates were 16.8% and 41.7% in the late toltrazuril-treated (at 84 h)and infected non-treated chickens, respectively. Lesions scores of necrotic enteritis or coccidiosis in infected,non-treated chickens were significantly more severe compared with negative controls (PB0.01) and latetoltrazuril-treated (at 84 h) chickens (PB0.05). In conclusion, application of toltrazuril before Eimeriachallenge protected chickens from coccidiosis and indirectly from successive necrotic enteritis caused byC. perfringens infection.

Introduction

Chicken necrotic enteritis (NE) is found worldwide andcauses high economic losses every year (Van der Sluis,2000; Lovland & Kaldhusdal, 2001). NE is caused byClostridium perfringens type A, a Gram-positive anae-robic spore-forming bacterium (Si et al., 2007). Twoforms of NE (clinical and mild, i.e. sub-clinical) havebeen described and linked with lesions in the liver andcholangiohepatitis (Kaldhusdal & Hofshagen, 1992;Songer, 1996; Gholamiandehkordi et al., 2007; Olkowskiet al., 2008). C. perfringens type A secretes different typesof toxins such as alpha, beta 2, necrotic enteritis B-liketoxin (NetB) and TpeL. For a long time, C. perfringensalpha toxin was suggested as the main virulence factor inNE development in poultry (Al-Sheikhly & Truscott,1977). Recently, expression of the pore-forming toxinNetB by C. perfringens has been described to be closelyrelated with NE occurrence in chickens (Keyburn et al.,2008; Savva et al., 2013) by destroying target cell mem-branes. Accordingly, netB-negative strains did not in-duce NE in experimental infection models (Keyburnet al., 2008, 2010). Chicken coccidiosis is the predispos-ing disease that is most commonly associated with NEand therefore co-infection models involving coccidia and

clostridia may be employed to induce NE lesions(Williams et al., 2003; Williams, 2005).

Coccidiosis is a protozoal disease causing depressedweight gain and reduced feed intake, and depending onthe respective Eimeria spp. a varying mortality rateoccurs (Williams, 1996). It has been shown to increasethe risk of NE occurrence (Al-Sheikhly & Al-Saieg,1980) by damage of epithelia (Williams, 2005), proteinleakage and increase of mucus secretion allowingC. perfringens to replicate and produce toxins (VanImmerseel et al., 2004, 2009; Collier et al., 2008; Cooper &Songer, 2010). NE was effectively prevented by mixingantimicrobial growth promoters in the broiler feed(Elwinger et al., 1998; Lanckriet et al., 2010). However,due to the emergence of bacterial resistance, the use ofantibiotics is and will be further diminished. Variousmethods for NE prophylaxis have been investigated;for example, treatment with ionophorous anticoccidialdrugs (Williams, 2005) or a so far experimental approachof vaccination with a C. perfringens toxoid vaccine (Motet al., 2013). The effect of anticoccidials in a subclinicalNE model was tested by Lanckriet et al. (2010) andnarasin, maduramicine and salinomycine showed a

*To whom correspondence should be addressed. Tel: �49 341 9738085. E-mail: bangoura@vetmed.uni-leipzig.de

Avian Pathology, 2013

http://dx.doi.org/10.1080/03079457.2013.823476

Received 11 June 2013

# 2013 Houghton Trust Ltd

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certain efficacy. Toltrazuril is an anticoccidial drug andeffective on all intracellular stages of Eimeria, especially1 to 168 h after infection (Laczay et al., 1995). In thisstudy, the influence of toltrazuril (Baycox†; Bayer VitalGmbH, Leverkusen, Germany) application on NE atvarious time points before and after coccidial infectionwas tested.

Materials and Methods

Eimeria strains. The Houghton strain of Eimeria tenella (Dr D. P. Blake,

Royal Veterinary College, University of London, Hatfield, UK) and an

Eimeria brunetti field isolate (Institute of Parasitology, Faculty of

Veterinary Medicine, Leipzig University) were passaged in susceptible

broiler chickens before use (data not shown). Oocysts were sterilized in

12% sodium hypochlorite and antibiotics (Raether et al., 1995) and

washed with phosphate-buffered saline (PBS, pH 7.4; Sigma Aldrich,

Taufkirchen, Germany) before storage in 2% potassium dichromate

at 48C. Oocysts were washed in PBS and the number of sporulated

oocysts was calculated by a modified McMaster method. To ensure the

purity of Eimeria strains, a sample of the inoculum containing both

E. tenella and E. brunetti was tested with multiplex polymerase chain

reaction (PCR) as described by Su et al. (2003) using species-specific

primers (Table 1). Also Eimeria acervulina, Eimeria maxima and

Eimeria necatrix were used as positive controls. Briefly, DNA was

extracted using a QIAamp DNA mini kit (Qiagen, Hilden, Germany).

The PCR was carried out in 25 ml reaction volume containing 2 mM

magnesium chloride, 0.5 mM of each primer, 1 mM dNTP and

0.5 u Taq polymerase (Promega, Mannheim, Germany) in the appro-

priate buffer. The cycler programme comprised one initial denaturation

cycle (948C for 45 sec), 35 amplification cycles (948C for 45 sec, 508C for

30 sec and 728C for 45 sec) and a final extension step (728C for 5 min).

PCR products were analysed by 2% agarose gel electrophoresis.

Clostridium perfringens. The C. perfringens strain (laboratory no. 2�288)

kindly provided by RIPAC-LABOR (Potsdam-Golm, Germany) was

used in this study. NetB toxin gene presence was determined by PCR

as described by Baums et al. (2004) (Table 1). C. perfringens strain was

cultured anaerobically at 378C on neomycin�polymyxin blood agar

(neomycin (100 mg/l; Carl Roth, Karlsruhe, Germany)/polymyxin B

(50 mg/l; Sigma-Aldrich, Buchs, Switzerland) agar for 24 h followed by

incubation in reinforced clostridial medium (Sifin GmbH, Berlin,

Germany) at 378C for 24 h. The solution was centrifuged (2500�g,

10 min) and the pellet was resuspended in PBS, pH 7.4. The bacterial

count was determined using the plating colonies method (Sanders, 2012).

Chickens and experimental design. Ninety-six 1-day-old commercial

broiler chickens (Cobb 500†; Cobb-Germany Avimex GmbH, Wiedemar,

Germany) were reared on the floor and fed feed free from antibiotics

and anticoccidials. Fourteen-day-old chickens were caged and divided

into eight groups (n�12), designated groups 1 to 8. Chickens of groups

1 to 6 were inoculated orally with 25,000 oocysts of E. tenella and

75,000 oocysts of E. brunetti at the age of 18 days. At the age of 22 days,

chickens of groups 1 to 6 were additionally infected by oral gavage with

109 colony-forming units (CFU) C. perfringens in 1 ml water. Chickens

of groups 1 to 5 were treated with 75 parts/106 toltrazuril (Baycox†) in

drinking water for 8 h on two consecutive days at different time points

in relation to the Eimeria infection (Table 2). The treatment started

exactly at the time point given in Table 2; that is, 12 h before infection

in group 1 and so on (at 22 h on the respective study day (SD)). Group 6

was not treated and served as a positive control. Chickens in groups

7 and 8 remained uninfected and served as controls, group 7 was treated

with toltrazuril at 17 and 18 days of age while group 8 provided the non-

treated negative control. Two sections were carried out in 24-day-old

and 30-day-old chickens for evaluation of coccidiosis and NE lesions.

Samples for histological examination were collected from 2 cm of each

jejunum, ileum and caecum in 10% phosphate buffered formalin. Blood

samples were collected after slaughter in citrate-treated tubes (Life

Technologies, Darmstadt, Germany), centrifuged at 2000�g for 10 min.

The plasma was stored at �208C until use.

Growth inhibition test of toltrazuril on C. perfringens. The minimal

inhibitory concentration (MIC) of toltrazuril on C. perfringens was

determined in triplicate in a 48-well microtitre plate. Then 50 ml

C. perfringens either exponential or stationary phase cultures (105 CFU/

ml) were added to 450 ml broth media (reinforced clostridial medium)

containing different concentrations of toltrazuril (500, 250, 125, 62.5, 31,

15, or 7 mg/ml, respectively). Plates were incubated anaerobically overnight

at 378C. The lowest concentration of toltrazuril that inhibited growth

of C. perfringens compared with unmedicated wells was indicated as the

MIC value.

Table 1. Primers used in this study.

Target sequence Primer Fragment length (base pair) Reference

E. acervulina (ITS-1) F: 5?-ctgcgagggaacgcttaat-3? 303 Su et al. (2003)R: 5?-aacgaacgcaataacacacg-3?

E. maxima (ITS-1) F: 5?-ttgtggggcatattgttgtg-3? 151 Su et al. (2003)

R: 5?-caatgaggcaccacatgtct-3?E. tenella (ITS-1) F: 5?-cgctgctggttttacaggtt-3? 463 Su et al. (2003)

R: 5?-gctgaagcaaagttccaagc-3?E. brunetti (ITS-1) F: 5?-agcttggattttcgctcaga-3? 395 Su et al. (2003)

R: 5?-cttccgtacgtcggatttgt-3?E. necatrix (ITS-1) F: 5?-gtcagtttttgcctgggtg-3? 385 Su et al. (2003)

R: 5?-acagaccgctacacaacacg-3?NetB toxin F: 5?-gctggtgctggaataaatgc-3? 383 Keyburn et al. (2008)

R: 5?-tcgccattgagtttccc-3?

F, forward; ITS-1, internal transcribed spacer 1; R, reverse.

Table 2. Experimental design.

Group

(n �12)

Eimeria spp.

infectiona

C. perfringens

infectionb

Toltrazuril treatment

(time point in relation to

Eimeria spp. infection)c

1 � � 12 h before infection

2 � � 12 h post infection

3 � � 36 h post infection

4 � � 60 h post infection

5 � � 84 h post infection

6 � � �7 � � 17 and 18 days old

8 � � �

aChickens inoculated orally at 18 days old with 25,000 oocysts

of E. tenella and 75,000 oocysts of E. brunetti. bChickens

inoculated orally at 22 days old with 109 CFU/ml C. perfringens.cChickens treated with 75 parts/106 toltrazuril in drinking water

for 8 h for two consecutive days; for groups 2 to 5 and 7, the time

point of the beginning of the treatment is given.

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Assessment of efficacy. Clinical signs, mortality and weight gain and feed

conversion ratio were noted. All chickens were observed clinically daily

throughout the experiment. Clinical signs and mortality were recorded.

For each group, fresh feed was weighed in and the residual feed was

weighed out daily. Body weight was assessed in 3-day intervals through-

out the study and the feed conversion ratio (FCR) was calculated.

Lesion scoring. Macroscopic lesions were scored separately for typical

coccidia-induced lesions according to Johnson & Reid (1970) and

for NE according to Prescott et al. (1978); Table 3) including scores

ranging from zero to four for both scoring systems (0 �no lesions;

4 �maximum lesion score). In addition, histological lesions were

assessed according to Gholamiandehkordi et al. (2007). In brief,

evaluation of histological lesions was based on the extent of villus

fusion, haemorrhages, epithelial defects and the amount of proteinac-

eous material in the gut lumen and done descriptively.

Oocyst counts in faecal samples. Faecal samples were examined for

Eimeria oocysts daily from 1 day before infection until the end of

the study using a McMaster counting chamber technique as described

by Long & Rowell (1958). Briefly, 100 g fresh faeces from each group

were collected daily and homogenized in 50 ml water. Six grams of the

homogenate were suspended in 60 ml saturated sodium chloride

solution (density: 1.2 g/l) and stirred for 2 min on a magnetic stirrer.

The suspension was used to fill six McMaster counting fields per

sample, if necessary after dilution. The oocyst content of the faecal

samples was calculated in terms of the oocyst content per gram of faeces

(mean count of six fields�100�dilution / 6). Additionally, faecal

samples were tested for presence of single Eimeria spp. using a multiplex

PCR as described above.

Re-isolation of C. perfringens. Intestinal contents (jejunum and caecum)

were tested for the C. perfringens at 24 and 30 days old. The bacterial

count was determined using the plating colonies method (Sanders, 2012)

on neomycin�polymyxin blood agar (neomycin (100 mg/l; Carl Roth)/

polymyxin B (50 mg/l; Sigma-Aldrich). C. perfringens colonies were

confirmed by matrix-assisted laser desorption/ionization time-of-flight

analysis (Shehata et al., 2013) to ensure specificity and the diagnosis and

to exclude misdiagnosis due to contaminating haemolysing bacteria.

Detection of E. tenella-specific antibody titres by enzyme-linked

immunosorbent assay. Antibodies against E. tenella were detected using

an enzyme-linked immunosorbent assay (ELISA) as described by

Constantinoiu et al. (2007). Briefly, a 96-well plate was coated with

100 mg/ml E. tenella oocyst protein in carbonate buffer (pH 9.6)

overnight. Free binding sites were blocked with 3% bovine serum

albumin in PBS with 0.05% Tween† 20 (PBST) for 2 h at room

temperature. Serum samples were diluted 1:100 in PBST and applied to

the plate for 1 h at room temperature. After washing (five times) with

PBST, the plates were incubated for 1 h with the secondary antibody

(1:8000 in PBS, horseradish peroxidase-labelled goat anti-chicken IgG;

Southern Biotech, Birmingham, AL, USA) followed by washing five

times. Substrate (3,3?,5,5?-tetramethylbenzidine) was added and the

reaction was stopped after 20 min by adding H2SO4 (1 M). The optical

density was determined in a plate reader at 450 nm. A standard curve

using a twofold dilution series (from 625 to 0.3 ng/ml) of unlabelled

chicken IgY (Southern Biotech) instead of antigen was used to calculate

the concentration (ng/ml) of antibodies. All measurements were done in

technical triplicate. The cut-off value was calculated based on the

nonspecific background levels of the negative control chickens of groups

7 and 8 using the following formula:

Cut�off ¼ mean value all negative serað Þþ3� standard deviation all negative serað Þ

Avidin ELISA. An ELISA plate (Corning Costar, Corning, NY, USA)

was coated with 100 ml/well of 2 mg/ml rabbit anti-avidin IgG antibodies

(Sigma-Aldrich) in sodium hydrogen carbonate buffer (NaHCO3;

Calbiochem, Nottingham, UK) and incubated for 1 h at room

temperature with shaking followed by washing twice with PBST.

Samples were diluted 1:50 and 1:100 in assay buffer (20 mM Tris/HCl

[pH 8.0], 0.15 M NaCl, 5 mM ethylenediamine tetraacetic acid, 0.1% [w/

v] bovine casein and 0.1% Tween 20 [v/v]). Test samples were analysed in

triplicate. Therefore, 100 ml diluted test sample was added to each well

and incubated for 1 h at room temperature with shaking. After washing

three times, diluted horseradish peroxidase-rabbit anti-avidin IgG

antibodies (1:1000 in assay buffer; Sigma-Aldrich) were added and the

plate was incubated for 1 h at room temperature. After washing three

times, the horseradish peroxidase activity was determined by adding 100

ml/well of 3 mM H2O2 and 1 mM 3,3?,5,5?-tetramethylbenzidine in 0.2

M citrate buffer (pH 4.0). The substrate reaction was stopped with 1 M

H2SO4 (50 ml/well). The optical density was measured with an ELISA

reader (Anthos htll, Anthos Labtec Instruments GmbH, Wals, Austria)

at 450 nm. The avidin concentration (ng/ml) was measured according to

a standard curve using 20 ng/ml to 0.15 ng/ml avidin (Kem-En-Tec

Diagnostic A/S, Copenhagen, Denmark; data not shown).

Statistical methods. All compiled data were analysed using IBM SPSS

Statistics 20† (IBM, New York, USA). All parameters were tested for

normal distribution by Kolmogorov�Smirnov test and analysed for

group differences by analysis of variance and subsequent Bonferroni

test (normally distributed data) or Kruskal�Wallis test and subsequent

Mann�Whitney U-test (non-normally distributed data), respectively.

Ethical approval. The trials were conducted in accordance with the

current European and national legal requirements. They were registered

and permitted by the responsible authority (Landesdirektion Leipzig,

file no. TVV13/12). Birds suffering from severe clinical signs exceeding

moderate clinical necrotic enteritis (e.g. showing sustained reluctance to

feed, apathy, immobility or other signs) were to be euthanized ahead of

schedule.

Results

PCR analysis of challenge strains. After passaging ofE. tenella and E. brunetti strains in susceptible commer-cial broiler chickens to be used for challenge, theywere tested with multiplex PCR. Both E. tenella andE. brunetti strains showed no contamination with otheroocysts as demonstrated for E. acervulina, E. maximaand E. necatrix (Figure 1a). Also, the toxin type ofC. perfringens tested with PCR showed the presence ofthe netB gene in the challenge strain (Figure 1b).

Growth inhibition test of toltrazuril on C. perfringens. TheMIC value for toltrazuril with reference to exponentialand stationary in vitro C. perfringens cultures was 62.5and 125 mg/ml, respectively.

Table 3. Lesion scoring for NE-typical lesions (based on

Prescott et al., 1978).

Lesion score

(NE) Observed macroscopic finding

0 No lesions

1 Small intestine wall grossly thinner than normal

and breaks or tears easily under mild tension but

has no gross evidence of mucosal necrosis or other

abnormalities

2 One or more necrotic foci of approximately 1 to 5

mm areas of full mucosal thickness

3 Necrosis more than 5 mm and consisting of

orange/brown necrotic debris

4 Large confluent necrotic areas of full mucosal

thickness in the small intestine affecting 25% or

more of the small intestine or birds that died from

intestinal lesions

Necrotic enteritis and coccidiosis 3

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Weight gain and feed conversion ration. Figure 2apresents the weight gain for all groups at 14, 24 and 30days of age. No significant difference was observedbetween infected and toltrazuril treated chickens (groups1 to 5) and negative control groups (groups 7 and 8)regarding body weight. However, infected non-treatedchickens (group 6) showed a significantly lower bodyweight (P B0.05) in 30-day-old chickens compared withall other groups (Figure 2a). A marked temporaryreduction of feed intake was observed only in the late-treated group 5 and the positive control group 6 for 4days starting on SD 22 (Figure 2b), and a slightdepression was shown on SD 23 in group 4. The othergroups did not show alterations in the feed intake curve(data not shown). Nonetheless, the positive controlgroup 6 broilers showed a higher FCR (P �0.05)compared with all other groups over the observationperiod (Figure 2c).

Clinical signs and mortality. Chickens infected withEimeria spp. and C. perfringens and treated early withtoltrazuril (groups 1 to 3) showed neither clinicalsigns nor mortalities during the observation period(Figure 3a). However, chickens infected and treated at60 h and 84 h (groups 4 and 5, respectively) showedclinical signs but less severe compared with infectedand non-treated chickens. The mortality rates were 8.3%(1/12) and 16.7% (2/12), respectively. Positive controlchickens (group 6) showed reduced feed intake, foamy tobloody diarrhoea and ruffled feathers approximately 36to 48 h after C. perfringens infection and showed amortality rate of 41.7% (5/12) within 2 weeks afterEimeria spp. infection (Figure 3a).

Eimeria spp. counting. Oocysts were not detected inchickens infected and treated early with toltrazuril(groups 1 to 3). Oocyst excretion (oocyst content pergram of faeces) in infected chickens treated at 60 h and84 h (groups 4 and 5, respectively) were significantly(P B0.001) lower compared with infected and non-treated chicken over all days of oocyst shedding (group6; Figure 3b). The examination of faecal samples for

different Eimeria spp. content using the multiplex PCR

revealed the presence of both E. tenella and E. brunetti in

the experimentally infected groups.

N P S

500

bp M E. tenella E. brunetti E. acervulina E. maxima E. necatrix

P S N P S N P S N P S N P S N

1000

500

100

(a) (b)

Figure 1. PCR analysis of challenge strains. 1a: PCR analysis of E. tenella and E. brunetti (P, positive control; S, E. tenella and

E. brunetti inoculum; N, negative control). 1b: NetB toxin-specific PCRs (N, negative control; P, netB-positive control; S, challenge strain).

0

20

40

60

80

100

120

140

SD (age in days)

Dai

ly f

eed

inta

ke (

g) group 1 group 4 group 5 group 6

(b)

(c)

0

200

400

600

800

1000

1200

Group number

Ave

rag

e b

od

y w

eig

ht

(g)

SD 14 SD 24 SD 30

*

(a)

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8Group number

FC

R

SD 24 SD 30

Figure 2. Effect of toltrazuril treatment on (2a) average body

weight, (2b) feed intake, and (2c) FCR. *Significant difference

(PB0.05) compared with negative controls on SD 30.

4 A. A. Alnassan et al.

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Re-isolation of C. perfringens. C. perfringens was reiso-lated from the intestinal content of all infected groups1 to 6 at both time points of examination (SD 24 and SD30) varying from 3 CFU/g (minimum, group 1) to 4.9�108

CFU/g (maximum, group 6) on SD 24 (Figure 3c) andfrom 31 CFU/g (minimum, group 1) to 6.3�104 CFU/g(maximum, group 5) on SD 30 for the jejunum, respec-tively. On SD 24, the number of C. perfringens CFU injejunal and caecal contents of positive control chickens(group 6) was significantly higher (P B0.05) comparedwith all treated groups for jejunum and for treated groups1, 4 and 5 for caeca (Figure 3c). The lowest intestinal C.perfringens counts were found in the prophylacticallytoltrazuril-treated group (group 1).

Pathological and histological lesions due to coccidiosisand necrotic enteritis. Chickens infected with Eimeria spp.and C. perfringens and treated early with toltrazuril(groups 1 to 3) showed no lesions typical for coccidiosis.NE typical lesions were of mild to moderate character ingroups 1 to 4 as given in Table 3. Only one bird fromgroup 4 and two birds from group 5 showed severe lesionsof score 4 due to NE (Figure 4). On the other hand, lesionscores according to NE and coccidiosis in infected, non-treated chickens (group 6) were significantly higher thanin all other groups (P B0.01 compared with negativecontrol groups 7 and 8, and P B0.05 compared withinfected-treated groups 1 to 5 (SD 24) or groups 1 to 4(SD 30), respectively; Table 4). The histopathologicalexamination revealed severe lesions in infected, non-treated group 6 chickens associated with massivecolonization of the mucosal villi with C. perfringens. Thevilli as well as the lamina propria showed severe destruc-tion and infiltration with monocytes and heterophils.(Figure 4). Mild pathological lesions were observed ininfected and treated chickens including infiltration ofheterophils, colonization of C. perfringens and moderatedesquamation of epithelial cells. No histological changeswere noted in negative control groups.

E. tenella-specific antibody titres. A cut-off value of7.67 ng/ml serum was calculated for the ELISA. Accord-ingly, 15 days after Eimeria spp. infection, E. tenella-specific seroconversion was detected in groups 2 to 6(Figure 5). Serotitres were significantly (P B0.05) higher

(a)

(b)

(c)

0

1

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7 8Group number

Mea

n c.

Per

frin

gen

s c

ount

s (lo

g10

cfu/

g)

jejunum

caeca

*†

Figure 3. 3a: Survival rates of chickens. 3b: Shedding of Eimeria

spp. oocysts (sum) expressed as log10 oocysts per gram of faeces

(OPG; *PB0.001 compared with all other groups, summarized

over the observation period after Eimeria spp. infection). 3c: C.

perfringens CFU in jejunal and caecal contents on SD 24.

*Significant differences (PB0.05) compared with treated groups

for jejunum; $significant differences (P B 0.05) compared with

treated groups 1, 4 and 5 for caecum.

Table 4. Lesion scores.

E. tenella lesionsa E. brunetti lesionsa NE lesionsb

Group 24 days old 30 days old 24 days old 30 days old 24 days old n �6 30 days old n �6

1 0 0 0 0 0.590.8 0.3390.8

2 0 0 0 0 0.6790.8 0.1790.4

3 0 0 0 0 0.6790.8 0.590.8

4 0 (0 to 1) 0 0 0 (0 to 0.25) 1.1791.3 0.590.8

5 0 (0 to 1) 0 0 (0 to 1) 0 (0 to 0.25) 1.1791.2 0.6791

6 3 (2.75 to 4)* 1 (0.75 to 1) 3 (2 to 3)* 0 (0 to 1) 3.6790.5** 1.591$

7 0 0 0 0 0 0

8 0 0 0 0 0 0

aScoring of Eimeria spp.-specific lesions according to Johnson & Reid (1970), median values and first/third quartile pooled for

sections (n �6 per group); score range, zero (no lesions) to four (extremely severe lesions). *P B0.05 compared with all infected and

treated groups 1 to 5, and P B0.01 compared with groups 7 and 8. bScoring of NE-specific lesions according to Prescott et al. (1978),

mean values and standard deviation (n �6 per group). **P B0.01 compared with groups 1 to 5; $P B0.05 compared with groups 1 to 4.

*P B0.05 compared with all infected and treated groups 1 to 5, and P B0.01 compared with groups 7 and 8.

Necrotic enteritis and coccidiosis 5

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in infected non-treated chickens (group 6) comparedwith all other groups.

Avidin serum concentration. Avidin serum levels wereincreased in all infected groups. In 24-day-old chickens(i.e. 6 days after Eimeria spp. infection), avidin levelswere significantly higher (P B0.01) in the positivecontrol group 6 than in all other groups (except group5 treated late at 84 h after Eimeria spp. infection). Avidinwas reduced in surviving chickens at the age of 30 dayscorresponding to 12 days after Eimeria spp. infection(Figure 6).

Discussion

The presence of C. perfringens in the intestinal tract ofbroiler chickens, even at high numbers, is not sufficientto produce NE (Long & Truscott, 1976; Cowen et al.,

1987; Kaldhusdal et al., 1999; Pedersen et al., 2003)

without an additional predisposing factor. Pre-exposure

to some Eimeria spp. has been strongly implicated

in promoting Clostridium-related diseases such as NE

(Williams et al., 2003; Lee et al., 2011). Therefore, we

investigated the preventive potential of toltrazuril

against NE due to simultaneous infection with E. tenella,

E. brunetti and C. perfringens in commercial broiler

chickens. In broilers, strategic medication with toltrazur-

il reduces coccidiosis cycling. Toltrazuril has been shown

to successfully control coccidiosis in broilers with just a

single 2-day treatment course (Mathis et al., 2003). To

our knowledge, there is only one report about resistance

of an Eimeria isolate against toltrazuril (Stephen et al.,

1997), although it is used regularly in the field. Thus, at

present, resistance of coccidia against this compound

does not appear to be a relevant factor in the poultry

industry in contrast to other anticoccidials like iono-

phors or quinolones in which resistances are widespread

(Vertommen et al., 1990). In a previous study in our

Figure 4. NE-typical lesions. 4a: uninfected and treated group 7, no NE lesions. 4b: focal necrosis in groups 4 and 5 treated late (60 h or

84 h after Eimeria spp. infection, respectively) 4c, 4d: macroscopic and histological lesions of severe NE (score 4) in positive control

group 6. Arrows, necrotic foci; arrowheads, cell detritus and exudate above destroyed mucosa.

0

5

10

15

20

25

30

35

40

1 2 3 4 5 6 7 8group number

anti

-Eim

eria

ten

ella

an

tib

od

yco

nce

ntr

atio

n (

ng

/ml)

cutoff=7.67 ng/ml

*

Figure 5. E. tenella-specific serotitres in 30-day-old chickens;

groups 1 to 6 were inoculated with Eimeria spp. *PB0.05

compared with all other groups; cut-off value marked by broken

line.

0

50

100

150

200

250

1 2 3 4 5 6 7 8group number

seru

m a

vid

in (

ng

/ml)

SD 24 SD 30*

Figure 6. Avidin serum concentration (ng/ml). *PB0.05 for

24-day-old chickens compared with all groups except group 5.

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laboratory, early treatment with toltrazuril preventedNE resulting from simultaneous infection with Isosporasuis and C. perfringens in newborn piglets (Mengel et al.,2012).

The efficacy of toltrazuril application at 12 h beforeEimeria infection as well as at 12 h, 36 h, 60 h and 84 hafter Eimeria infection was compared. Early treatmentof toltrazuril up to 36 h after Eimeria infectioneffectively protected chickens from both coccidiosisand severe NE. Late-treated groups (60 h or 84 h afterEimeria infection) showed higher FCR and mortalitiesoccurred. The highest mortality rate (41.7%) was ob-served in positive control chickens and the lesions ofNE and coccidia were most pronounced. This severity ofclinical disease is caused by simultaneous infection withthe pathogens E. tenella, E. brunetti and C. perfringens.In a previous investigation in this model, the sameinfection doses of E. tenella and E. brunetti orC. perfringens, respectively, did not induce any mortalityin broilers if administered separately (data not shown).The MIC test performed for toltrazuril revealedan intrinsic antibacterial effect of the compound onC. perfringens that, to the best of our knowledge, was notdescribed before. Nonetheless, the inhibitory concentra-tion for exponential cultures was 125 mg/ml in vitro,which is above the administered dosage of 75 mg/ml indrinking water. In addition, in vivo a variety of influen-cing factors such as dilution by ingesta, contact time,absorption by the gut, metabolism, and others have to beconsidered. Thus we suspect that the early treatmentwith toltrazuril that took place before C. perfringensinfection at least mainly inhibited C. perfringens growthindirectly by suppression of intracellular parasite repli-cation and epithelial cell damage. This assumption iscorroborated by the fact that earlier toltrazuril treat-ments were more effective against NE than later treat-ments which suggest a higher chance of direct contactbetween C. perfringens and toltrazuril. In the presentstudy no Eimeria oocysts were detected after earlytreatment with toltrazuril before or up to 36 h afterEimeria infection, respectively. Similar results wereachieved by Mengel et al. (2012), who applied a singledose of toltrazuril and completely suppressed oocystexcretion and diarrhoea in piglets co-infected with I. suisand C. perfringens. Mengel et al. (2012) also concludedthat toltrazuril prevents a severe coccidial multiplica-tion and thus increased mucus production, which wouldpromote a secondary clostridial infection and clinicaldisease.

In the present study, infected and late toltrazuril-treated chickens (60 h and 84 h after Eimeria infection)and positive control chickens showed C. perfringens inintestinal contents, whereas it could be rarely reisolatedfrom early toltrazuril-treated chickens. This correspondswith previous observations which state that the intestineof birds suffering from necrotic enteritis contains largenumbers of C. perfringens organisms, whereas in healthybroilers much lower or even zero counts are normal,especially in the jejunum (Long et al., 1974; Baba et al.,1997; Si et al., 2007).

Avidin is a nonspecific acute phase protein, a markerto inflammation and bacterial infections (Kunnas et al.,1992; Matulova et al., 2012). Avidin was significantly(P B0.05) increased in all infected chickens 6 days afterEimeria infection compared with the negative controlgroups and it was significantly higher (P B0.05) in

positive control chickens compared with early toltrazur-il-treated chickens. This corresponds to the more severeintestinal lesions and inflammation seen in the untreatedbirds (Figure 4).The background avidin level displayedby all chickens including the control groups is regarded asnormal since avidin is a defence protein induced by manydifferent stress factors (Tuohimaa et al., 1989). Interest-ingly, the highest avidin levels that were measured co-incided with the most severe gut lesions, and as soon asthe intestinal inflammation decreased the avidin levelsdecreased as well within only a few days, which shows theacute marker properties in this disease.

Humoral response reflects stimulation of the immunesystem against Eimeria infections in poultry (Wallach,2010) and antibody titres may thus be used as anindicator for immunological stimulation althoughantibody levels do not necessarily correspond directlywith the effectivity of the anticoccidial immune reac-tion involving the cellular response. In the present trial,E. tenella antibodies were detected in the groups thatwere treated after Eimeria spp. infection (metaphylacti-cally) or remained untreated. Prophylactic anticoccidialtreatment that prevented the initial parasite replicationalso resulted in antibody levels slightly under the cut-offvalue and did not allow development at least of asignificant humoral immune reaction. At the same time,this proves an immunological reaction of chickens alsoto the early asexual parasitic stages inhibited by earlymetaphylactic toltrazuril treatment, thus confirming thatabrogation of the Eimeria lifecycle by toltrazuril doesnot impair the immune response (Greif, 2000). This is inagreement with early findings in E. tenella-infectedchickens where sulphonamide treatment affected theendogenous parasite stages without inhibiting the im-mune response (Horton-Smith & Taylor, 1945; Kendall &McCullough, 1952). Nonetheless, Shirley et al. (1986)observed normal immunogenicity in a precociousE. brunetti strain with a shortened asexual developmentindicating high immune response to sexual Eimeriastages, which explains the significantly higher antibodytitres observed in the infected untreated birds.

In conclusion, toltrazuril medication helped to reducesevere NE in the tested model by reducing coccidiallesions that predispose the host to C. perfringens infec-tion. Under consideration of the withdrawal period,toltrazuril or other anticoccidial drugs may be applied inthe anticipated time frame of infections with differentEimeria spp. under intensive rearing conditions; that is,in 15-day-old to 16-day-old broilers (Morris et al., 2007).Prophylactic treatment (i.e. before infection) is effectivebut impairs the immune response. Alternatively, antic-occidial vaccines may be applied to prevent severe NEwithout the risk of formation of drug resistance;respective studies are pending.

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