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Veterinary Immunology and Immunopathology 122 (2008) 104–115

Coccidia-induced mucogenesis promotes the onset of necrotic

enteritis by supporting Clostridium perfringens growth

C.T. Collier a, C.L. Hofacre e, A.M. Payne f, D.B. Anderson f, P. Kaiser g,R.I. Mackie a,c,d, H.R. Gaskins a,b,c,d,*

a Department of Animal Sciences, University of Illinois at Urbana-Champaign, United Statesb Department of Pathobiology, University of Illinois at Urbana-Champaign, United States

c Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, United Statesd Institute for Genomic Biology, University of Illinois at Urbana-Champaign, United States

e Poultry Diagnostic and Research Center, Department of Avian Medicine, University of Georgia, Athens, GA, United Statesf Elanco Animal Health, Research and Development, Greenfield, IN, United States

g Institute for Animal Health, Compton, Berkshire, United Kingdom

Received 16 August 2007; received in revised form 12 October 2007; accepted 23 October 2007

Abstract

This study tested the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic

enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and

subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore

narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E.

acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment

on host immune responses and microbial community structure were evaluated with histochemical-, cultivation- and molecular-

based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic

bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent increase in intestinal NE

lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal

interleukin (IL)-4, IL-10 and IFN-g RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, and goblet

cell number and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of

mucolytic bacteria and C. perfringens as well as a toxin production was greatest in EAM/CP-infected birds. The ionophore narasin,

which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-g expression, the growth of mucolytic bacteria

including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens.

Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth

advantage to C. perfringens, the causative agent of NE.

# 2007 Elsevier B.V. All rights reserved.

Keywords: Clostridium perfringens; Coccidia; Mucus; Necrotic enteritis

Abbreviations: CFU, colony forming unit; CP, Clostridium perfringens; DGGE, denaturant gradient gel electrophoresis; EAM, Eimeria

acervulina and maxima; HID/AB, high iron diamine/alcian blue; IFN, interferon; IL, interleukin; NAR, narasin; NE, necrotic enteritis; NIF, non-

infected; qPCR, quantitative polymerase chain reaction.

* Corresponding author at: University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801, United States.

Tel.: +1 217 244 3165; fax: +1 217 333 8286.

E-mail address: hgaskins@uiuc.edu (H.R. Gaskins).

0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetimm.2007.10.014

C.T. Collier et al. / Veterinary Immunology and Immunopathology 122 (2008) 104–115 105

1. Introduction

Necrotic enteritis (NE) is a small intestinal inflam-

matory disorder of economic significance (globally

greater than 2 billion US dollars/year) that primarily

affects neonatal chickens. The causative agent of NE is

Clostridium perfringens, a low GC, Gram-positive,

anaerobic, spore-forming bacterium that is found

commonly in soil, sewage, and the gastrointestinal tract

of animals and humans (McDonel, 1980; Shane et al.,

1984; Lindsay, 1996). Risk factors for the onset of

clinical disease include concurrent coccidial infection,

the removal of antibiotic growth promoters, and the

inclusion of diet components such as wheat and barley,

which are high in water-soluble non-starch polysacchar-

ides (Truscott and Al-Sheikhly, 1977; Shane et al., 1984;

Riddell and Kong, 1992; Ficken and Wages, 1997;

Langhout, 1998; Caplan and Jilling, 2001; Craven et al.,

2001). Necrotic enteritis shares strikingly similar

pathophysiological features with necrotizing enteroco-

litis (NEC), one of the most common and serious

gastrointestinal disorders of newborn infants (Gibbs

et al., 2007). NEC is responsible for the death of

approximately 1000 infants per year, which is compar-

able to the number of children under 15 years of age who

die of leukemia each year in the U.S (Gibbs et al., 2007).

Preterm neonates, which are often fed parenterally, are at

greatest risk for the development of NEC (Heird and

Gomez, 1994, 1996; Yeo, 2006). Although the etiology of

NEC is unknown, intestinal inflammatory cascades

associated with parenteral nutrition and the transition

from parenteral to enteral support possibly contribute to

the onset of the disorder (Heird and Gomez, 1996). In

previous work with a piglet model of total parental

nutrition (TPN), we observed an association between

small intestinal inflammation and mucogenesis (Conour

et al., 2002; Ganessunker et al., 1999). Enhanced mucus

production in the TPN piglet model was associated with

the selection of C. perfringens, which led to discovery of

the mucolytic nature of this opportunistic pathogen

(Deplancke et al., 2002).

The present study used a previously established

chick model of NE (Hofacre et al., 1998) to test the

hypothesis that the host inflammatory response to

eliminate intestinal coccidia results in increased mucus

production, and that this provides a growth advantage

for C. perfringens due to its ability to utilize mucus as a

substrate (Deplancke et al., 2002). In contrast, the direct

suppression of coccidia with an ionophore would

preclude a characteristic mucogenic response of the

host, thereby eliminating conditions favorable for C.

perfringens growth. Thus, we hypothesize that C.

perfringens growth on mucus represents an initial and

previously unrecognized stage of virulence, and that

this mode of growth may explain, in part, why

coccidiosis predisposes to NE.

2. Materials and methods

2.1. Experimental animals and infection model

Cobb male chicks (Cobb-Vantress Hatchery, Cleve-

land, GA) were distributed over three tier battery cages

in a conventional housing system. Five groups of birds

(42 birds/group) were randomly allotted to five

experimental treatments to test the main effect of

coccidiosis (non-infected (NIF) vs. infection with field

strains of Eimera vs. ionophore prophylaxis; Table 1).

Birds were housed in separate batteries according to

coccidia treatment status to avoid cross contamination.

Birds were fed a corn/soy-based diet throughout the

study, which was supplemented with 26% fishmeal

through 14 day of age (Hofacre et al., 1998). Birds

treated with narasin (Elanco Animal Health, Greenfield,

IN) were dosed from days 1 to 28 of the trial (100 ppm/

kg feed). Narasin is a monovalent polyether ionophore

that is a fermentation product of Streptomyces

aureofaciens. It is used in meal or pellet feed as an

aid in the prevention of coccidiosis and NE in broiler

chickens (Harris et al., 1998). Birds were inoculated by

oral gavage at 14-day of age with field isolates of

Eimeria acervulina (75,000 oocysts per bird) and E.

maxima (20,000 oocysts per bird; EAM, EAM/CP and

NAR/EAM/CP), which colonize the small and large

intestine, respectively. On days 18, 19 and 20, all birds

(except NIF and EAM treatment groups) were orally

gavaged with a broth culture of a field isolate of C.

perfringens type A (108 CFU/ml, 1 ml per bird; CP,

EAM/CP and NAR/EAM/CP; Hofacre et al., 1998).

Baseline values were determined prior to the onset of

NE at day 20 by randomly selecting birds (14 birds/

(treatment day)) to be euthanized for lesion scores and

to collect liver and intestinal samples for microbial and

pathophysiological assays described below. Based on

previous studies sampling was subsequently performed

at the peak of infection on day 22 when the symptoms of

NE or most severe and day 28 when recovery has

occurred (Hofacre et al., 1998; Collier et al., 2003).

2.2. Intestinal barrier function, lesions and

mortality

Barrier function of the intestinal mucosa was

determined by the indirect method of bacterial

C.T. Collier et al. / Veterinary Immunology and Immunopathology 122 (2008) 104–115106

Table 1

Summary of intestinal mucosal barrier functiona

Aerobic bacteria (average CFU/g) Anaerobic bacteria (average CFU/g) Clostridium perfringens

(average CFU/g)

Day 20 Day 22 Day 28 Day 20 Day 22 Day 28 Day 20 Day 22 Day 28

Liver

NIF 0.0 21.1 0.8 36.8 1.1 0.4 0.3 0.0 0.0

EAM 0.0 0.7 2.0 34.7 8.1 0.8 0.7 0.3 0.0

CP 0.0 2.5 1.1 55.1 16.3 0.0 0.4 0.0 0.0

EAM/CP 0.0 1.1 0.3 38.0 1.9 0.2 0.7 0.0 0.0

NAR/EAM/CP 0.0 4.3 0.1 29.2 3.8 0.1 1.5 0.1 0.0

Spleen

NIF 164.9 2.5 a 2.4 139.9 a 7.6 3.8 0.2 a 0.0 0

EAM 764.2 120.8 b 38.8 614.2 a,b 287.1 36.7 5.3 a,b 69.5 0.1

CP 371.1 10.7 a,b 12.0 424.6 a 12.3 6.7 3.7 a,b 0.0 0

EAM/CP 622.8 6.5 a,b 68.5 1531.7 b 3.3 41.0 15.5 b 0.0 0

NAR/EAM/CP 347.2 8.1 a,b 3.4 412.6 a 2.2 1.9 3.9 a,b 0.0 0

NIF: non-infected; EAM: inoculated with Eimeria acervulina and E. maxima; CP: C. perfringens; EAM/CP: EAM and CP; NAR/EAM/CP: narasin,

EAM and CP. Means with different letters within day and tissue are significantly different ( p < 0.05).a Determined by the translocation of aerobic bacteria, anaerobic bacteria, and C. perfringens to the liver or spleen at days 20, 22 and 28 (n = 7/

(treatment day)) and standardized to respective organ weight (g).

translocation to liver and spleen (Filos et al., 2004).

Total aerobic and anaerobic bacteria were quantified

from liver and spleen by standard aerobic and anaerobic

cultivation techniques on mixed media agar plates and

blood agar plates for C. perfringens growth. Values are

expressed as the average number of bacterial CFUs

standardized to organ weight, recovered per chick in

each treatment. Coccidial and necrotic lesions in the

jejunum and ileum were assessed and scored in 14

randomly selected birds per treatment at days 20, 22

and 28. Necrotic enteritis lesion scoring was based on a

0–3 score, with 0 being normal and 3 being the most

severe as previously described (Collier et al., 2003).

The jejunum and ileum were scored for coccidiosis

lesions using the system of Johnson and Reid (1970),

wherein 0 is normal and 1, 2, 3, or 4 indicate increasing

severity of infection. Data are presented as the

Table 2

Summary of coccidial and NE lesions and chick mortality

Treatments Coccidial lesionsa

NIF 0.0 a

EAM 86.0 c

CP 7.0 a

EAM/CP 86.0 c

NAR/EAM/CP 14.0 b

NIF: non-infected; EAM: inoculated with E. acervulina and E. maxima; CP: C

and CP. Means with different letters are significantly different ( p < 0.05).a Percentage of birds (n = 14/treatment) exhibiting coccidial lesions in jeb Percentage of birds (n = 14/treatment) exhibiting NE lesions in ileum ac Cumulative mortality in chicks (n = 42/treatment) from days 0 to 28

per battery.

percentage of animals exhibiting coccidial or NE

lesions. The number of bird deaths attributable to NE

was assessed cumulatively from day 0 to 28 of the trial

and averaged across batteries within day for statistical

analysis.

2.3. Chicken cytokine and intestinal mucin RNA

expression

Interleukin (IL)-4, IL-10, and interferon (IFN)-g

RNA expression was quantified from ileal tissue

samples via a real-time PCR assay as described

previously with the following modifications (Rothwell

et al., 2004). Template RNA was reverse transcribed

and relative expression of mRNA transcripts was

measured in triplicate via SYBR Green PCR amplifica-

tion in a GeneAmp 5700 Sequence Detection System

Necrotic lesionsb Bird mortalityc

0.0 a 0.0 a

7.0 a 0.0 a

7.0 a 1.2 b

29.0 b 3.4 c

0.0 a 0.5 a

. perfringens; EAM/CP: EAM and CP; NAR/EAM/CP: narasin, EAM

junum at day 22.

t day 22.

due to necrotic enteritis. Values represent average bird mortality

C.T. Collier et al. / Veterinary Immunology and Immunopathology 122 (2008) 104–115 107

(Applied Biosystems, Foster City, CA; Collier et al.,

2003). Chicken intestinal mucin RNA expression was

similarly quantified from ileal tissue as described

previously (Smirnov et al., 2004). A standard curve was

generated with host-derived RNA for all primer sets

and optimal sample dilution rates determined via an

initial serial dilution of one sample from each of the

treatments (1/10, 1/100, and 1/1000 dilution). Samples

were then assayed for each primer set and, after

normalization to 28S rRNA expression, are presented

as fold change in total number of cycles (40 cycles)

minus the threshold cycle value (Ct), the cycle at which

Fig. 1. Quantitative expression of IL-4 (a), IL-10 (b), IFN-g (c) and chicken

22 and 28 in the ileum presented as fold change in 40 � Ct values in chicks (n

Eimeria acervulina and E. maxima (EAM); Clostridium perfringens (CP);

Values not sharing a common letter within day are different ( p < 0.05).

the change in the reporter dye passes a significance

threshold (40 � Ct).

2.4. Goblet cell histology

Goblet cell number and size of the mucus-containing

theca per cell were quantified in the ileum as indicators

of intestinal mucogenesis for chicks sampled on day 22

(time of most severe infection) and day 28 (end stage of

infection). Goblet cells where stained with high iron

diamine/alcian blue (HID/AB) to distinguish those

synthesizing sialo- and sulfomucins, the two major

intestinal mucin (MUC) RNA (d) normalized to 28S rRNA at days 20,

= 7/treatment) that were either non-infected (NIF); or inoculated with

EAM and CP (EAM/CP); or narasin, EAM and CP (NAR/EAM/CP).

C.T. Collier et al. / Veterinary Immunology and Immunopathology 122 (2008) 104–115108

C.T. Collier et al. / Veterinary Immunology and Immunopathology 122 (2008) 104–115 109

types of acidomucins (Kandori et al., 1996; Deplancke

and Gaskins, 2001). Sulfo and sialomucin-positive

goblet cells were quantified from three intact villi and

surrounding crypts per sample as described previously

(Ishikawa et al., 1989; Enss et al., 1992; Kandori et al.,

1996; Conour et al., 2002). All samples were standar-

dized to the corresponding surface area (mm2) of each

villus or crypt. Adobe Photoshop 7.0 was used to measure

the length and width of the goblet cell theca in pixels, as

analyzed at 400�magnification (screen resolution set to

680 � 480 total pixels). Fifty goblet cells were analyzed

per bird (350 goblet cells per treatment). This was

performed for both ileal crypts and villi.

2.5. Community structure of the ileal microbiota

Ileal bacterial profiles were determined by 16S-V3

rDNA PCR-DGGE as described previously (Muyzer

and Smalla, 1998; McCracken et al., 2001). Discovery

Series Diversity Database software (Bio-Rad; version

2.1) was used to analyze banding patterns by measuring

the migration distance and intensity of the bands within

each gel lane. Ward’s algorithm was used to construct a

dendrogram of bacterial community structure as

described previously (Muyzer and Smalla, 1998;

Simpson et al., 1999) and presented in the Diversity

Database manual (Bio-Rad).

2.6. Bacterial mucolysis

Growth of mucolytic bacteria was analyzed via a

mucolysis assay developed by Deplancke et al. (2002) to

evaluate possible mechanisms that provide C. perfrin-

gens a selective growth advantage in the intestine. The

number of colonies that grew on mucin-limiting medium

(composition defined in Deplancke et al., 2002) is

expressed as a percentage of the 20 colonies per bird

collected from the habitat-simulating medium

(Deplancke et al., 2002). Additionally, the mucolytic

potential of 20 colonies selected from pure C. perfringens

cultures was examined in the same medium.

2.7. Quantification of C. perfringens growth and a

toxin expression

The colonization of C. perfringens and associated

a toxin RNA transcripts were analyzed via real-

Fig. 2. Representative photomicrographs (400�) of ileal epithelium of (a) N

mark goblet cells stained with HID/AB. Images on the right are representative

for quantitative analysis of treatment effects. Quantitative evaluation of muco

in (c) crypts and (d) villi and the percentage of goblet cells per surface area (

either non-infected (NIF); or inoculated with E. acervulina and E. maxima (E

and CP (NAR/EAM/CP). Values not sharing a common letter are different

time quantitative PCR (qPCR) as described pre-

viously (Wang et al., 1994; Deplancke et al.,

2002; Baums et al., 2004) in a GeneAmp 5700

Sequence Detection System. All transcripts were

validated and standardized using 1/10, 1/100,

and 1/1000 serial dilutions of RNA (250 mg/ml)

isolated from a pure C. perfringens culture. All

standards and unknowns from mucosal and luminal

contents from the ileum are expressed as the average

percentage of C. perfringens rDNA relative to total

bacteria rDNA.

2.8. Statistics

The occurrence of necrotic and coccidial lesions,

intestinal barrier function, goblet cell number and

theca size, qPCR data, mucolytic potential and

mortality were subjected to analysis of variance

(ANOVA) using SAS (Version 6.09; The SAS Institute,

Cary, NC). The partitioned sources of variation

included treatment, day and their interactions. Specific

treatment comparisons were made using Fisher’s

Protected Least Significant Difference test with an

assigned p-value of <0.05. Dendrograms of clustering

patterns of microbial profiles were generated with

Diversity Database (Version 2.2.0; BioRad) using

Ward’s algorithm (McCracken et al., 2001; Collier

et al., 2003). Pathological indices and data derived

from molecular- and culture-based techniques were

evaluated by Spearman correlation analysis using

SAS, to evaluate their correlation with the onset of

necrotic enteritis.

3. Results

3.1. Infection (EAM/CP) compromised intestinal

barrier function and increased intestinal lesions

and bird mortality

Intestinal mucosa barrier function data are presented

in Table 1 as the translocation of bacteria to the liver

or spleen. At day 20, aerobic and anaerobic bacterial

and C. perfringens CFUs were observed in the spleen

for all birds independent of treatment. This outcome

possibly reflects the immaturity of barrier function at

this early age. Despite substantial bird-to-bird variation

IF and (b) EAM/CP-infected birds. Scale bars are 10 mm and arrows

of A (NIF) and B (EAM/CP) and demonstrate goblet cell theca as used

genesis in the ileum at day 22 presented as goblet cell theca area (mm2)

10 mm2) in (e) crypts and (f) villi in chicks (n = 7/treatment) that were

AM); C. perfringens (CP); EAM and CP (EAM/CP); or narasin, EAM

( p < 0.05).

C.T. Collier et al. / Veterinary Immunology and Immunopathology 122 (2008) 104–115110

for all treatments, anaerobic bacterial and C. perfrin-

gens CFU/g were greater ( p < 0.05) in EAM/CP-

treated birds relative to control birds (NIF) at day 20. At

day 22, aerobic bacterial growth in the spleen was

greatest ( p < 0.05) in EAM-infected relative to all

other birds.

The number of coccidial lesions was greatest in

EAM- and EAM/CP-infected birds at days 20 (data not

shown) and 22 (Table 2). Differences in coccidial

lesions were not observed at day 28 (data not shown).

Necrotic enteritis lesions were greatest ( p < 0.05) in

the EAM/CP-treated group (Table 2) at day 22.

Necrotic lesions were observed in all treatment groups

at day 28 (data not shown). The low level of coccidial

and NE lesions in birds infected singularly with EAM

or CP presumably reflects the fact that both coccidia

and C. perfringens are ubiquitous in poultry (Lindsay,

1996; Collins, 1997). The severity of the coccidial and

NE lesion scores (based on the respective numerical

scoring systems) did not differ among the infection

treatments throughout the duration of the study (data

not shown).

Bird mortality was greatest in EAM/CP-treated birds

and not observed for NIF or EAM-treated chicks

(Table 2).

3.2. Infection (EAM/CP) increased inflammatory

cytokine and chicken intestinal mucin gene

expression

The expression of IL-4 RNA did not differ among

treatments at day 20 or 28, but was greater in all

infection treatments compared to the control birds

(NIF) at day 22 (Fig. 1a). Interleukin 10 RNA

expression was greatest ( p < 0.05) in the EAM/CP

treatment group at days 20 and 22, while expression in

EAM- and NAR/EAM/CP-treated birds was greater

( p < 0.05) than NIF and CP-treated groups at day 22

(Fig. 1b). Expression levels of IL-10 were similar

among treatments at day 28. At days 20 and 22, IFN-g

RNA expression was greatest ( p < 0.05) in EAM/CP-

treated birds (Fig. 1c). At day 28, IFN-g RNA

expression was suppressed and did not differ among

treatments. At day 20, mucin RNA expression was

greater ( p < 0.05) in all infected groups relative to

control birds, with the relative level of expression being

similar among infection treatment groups (Fig. 1d). Only

birds infected with C. perfringens without narasin (CP

and EAM/CP) exhibited greater ( p < 0.05) intestinal

mucin RNA expression compared to control (NIF) birds

at day 22. Ileal mucin RNA expression was similar

among all treatments at day 28.

3.3. Infection (EAM/CP) induced a mucogenic

response

Mucin in mature goblet cells accumulates in

membrane-bound granules, distending the apical region

(theca) of the cell. The size of the theca thus reflects

cellular mucus accumulation and was used as a measure

of mucogenesis (Fig. 2a and b). At peak inflammation

(day 22) in crypts, ileal goblet cell theca area was

greatest ( p < 0.05) in EAM-infected birds (Fig. 2c).

Average theca size of villus-associated goblet cells was

greatest ( p < 0.05) in EAM/CP-infected birds while

goblet cell theca sizes were larger ( p < 0.05) in NAR/

EAM/CP-treated birds than EAM-infected and NIF

birds (Fig. 2d). Although the number of sialo- or

sulfomucin goblet cells did not differ in villi or crypts of

the ileum, treatment differences were detected in the

number of total acidomucin-positive (sialo- and

sulfomucin-combined) goblet cells. At day 22 (peak

inflammation), the number of crypt-associated goblet

cells was greatest ( p < 0.05) in CP- and EAM/CP-

treated birds (Fig. 2e) while villus goblet cell numbers

were similar among treatments at day 22 (Fig. 2f).

Treatment differences in theca size and goblet cell

number were not observed at day 28 (data not shown).

3.4. Coccidial infection facilitated enrichment of

mucolytic bacteria including C. perfringens growth

and a toxin production

Bacterial profiles of the ileal microbiota of chicks at

days 20, 22, and 28 were determined via 16S-V3 rDNA

PCR-DGGE analysis (day 22, Fig. 3a). Treatment-

specific clusters were visually observed for all 3 days

and validated when banding patterns were compared via

dendrogram construction using Ward’s cluster algo-

rithm (day 22, Fig. 3b). These data indicate that

bacterial community structure was similar among birds

within treatment, but differed among treatments.

Mucolytic activity was determined for bacterial

cultures derived from ileal mucosal scrapings (n = 7

chicks/treatment) at days 20 and 22 (Fig. 4a). These

results demonstrate that mucolytic bacteria were in

greatest abundance ( p < 0.05) in EAM/CP-treated

birds relative to NIF, EAM- and NAR/EAM/CP-treated

birds at days 20 and 22. C. perfringens concentrations

were greatest in EAM/CP-treated birds relative to all

other treatments at day 22. The qPCR data further

demonstrate that the concentration of C. perfringens

was similar for NAR/EAM/CP and NIF birds, but

was significantly lower in these groups relative to all

other treatments at days 20 and 22 (Fig. 4b). The density

C.T. Collier et al. / Veterinary Immunology and Immunopathology 122 (2008) 104–115 111

Fig. 3. (a) Denaturing gradient gel electrophoresis (DGGE) profile generated from polymerase chain reaction (PCR) amplified V3-16S rDNA and (b)

resultant dendrogram from day 22 ileal lumen contents of chicks (n = 7/treatment) that were either non-infected (NIF); or inoculated with E. acervulina

and E. maxima (EAM); C. perfringens (CP); EAM and CP (EAM/CP); narasin, EAM and CP (NAR/EAM/CP). Each lane represents one bird; L:

reference ladder. Marked bands (arrow) in the reference ladders indicate the migration distance of the band corresponding to C. perfringens. The

dendrogram was constructed using Ward’s algorithm and Diversity Database software as described in Section 2. Clusters of similar treatments are boxed.

of C. perfringens was diminished in all samples by day

28 and treatment effects were not observed. The

expression of a toxin was detected only in CP and

EAM/CP samples at day 22 (data not shown). This

observation is consistent with C. perfringens concentra-

tion data at day 22.

3.5. Correlation between the onset of NE with

parameters indicative of a mucogenic response and

selection of mucolytic bacteria including C.

perfringens

Variables that were significantly correlated indepen-

dent of treatment are presented in Table 3. Inoculation

with EAM and CP (EAM/CP; NE infection model)

correlated positively to increased mortality (r = 0.87;

p < 0.01), occurrence of coccidial lesions (r = 0.92;

p < 0.01), necrotic lesions (r = 0.87; p < 0.01), IL-10

(r = 0.88; p < 0.05) and IFN-g (r = 0.87; p < 0.01)

RNA expression and crypt goblet cell numbers

(r = 0.86; p < 0.01). Infection with EAM/CP also

correlated positively with bacterial-related parameters

including C. perfringens growth (r = 0.91; P < 0.01), a

toxin production (r = 0.95; p < 0.01), and the density of

mucolytic bacteria (r = 0.89; p < 0.01). Conversely,

none of the variables were correlated (positively or

negatively) in NAR/EAM/CP-treated or control (NIF)

birds.

C.T. Collier et al. / Veterinary Immunology and Immunopathology 122 (2008) 104–115112

Fig. 4. (a) Percentage of mucolytic bacteria present in ileal mucosa of 20- and 22-day-old chicks and (b) the concentration of C. perfringens in the

ileal lumen at days 20, 22 and 28 expressed as a percentage of total bacteria 16S rDNA in chicks (n = 7/treatment) that were either non-infected

(NIF); or inoculated with E. acervulina and E. maxima (EAM); C. perfringens (CP); EAM and CP (EAM/CP); narasin, EAM and CP (NAR/EAM/

CP). Values not sharing a common letter are different ( p < 0.05).

4. Discussion

The present results support the hypothesis that

coccidial infection predisposes to NE through the

induction of local T cell-mediated inflammatory

responses that enhance intestinal mucogenesis. This

outcome was associated with alterations in the

composition of the ileal microbiota and enhanced

growth of the mucolytic bacterium C. perfringens, the

causative agent of NE. While there is considerable

evidence for intestinal goblet cell responsiveness to

inflammatory mediators, the extent to which microbial

communities are altered by intestinal mucogenesis has

received limited attention. Similarly, molecular

mechanisms underlying inflammatory responses to

coccidia are not well described and whether attendant

mucogenesis contributes to NE is unknown.

A possible link between coccidial-induced muco-

genesis and the onset of NE was conceived based on the

work of Ganessunker et al. (1999), Deplancke et al.

(2002) and Conour et al. (2002) with a piglet model of

TPN. This mode of nutrition induced small intestinal

inflammation including expansion of acidomucin goblet

cells along with a corresponding increase in the density

of mucolytic bacteria including C. perfringens.

Together, those data indicated the exquisite sensitivity

of intestinal goblet cells to inflammatory insults and the

likely impact of an intestinal mucogenic response on

microbial populations.

Similar to TPN conditions, intestinal coccidial

infection in the chicken is characterized by an

inflammatory response and results in increased water

and mucus content in fecal material, as well as diarrhea

(Shirley, 1997; Allen and Fetterer, 2002; Morris and

Gasser, 2006). The typical Eimeria infection is complex

as it involves both intracellular and extracellular stages

resulting in the rupture of infected host cells as the

pathogen propagates (Lillehoj and Trout, 1996;

McDougald, 1998). While mucogenesis is an inherent

component of host responses to a range of environ-

mental insults (Rogers, 1994; Kim et al., 1997; Meslin

et al., 1999), this phenomenon is best understood in the

context of inflammatory disorders of airway epithelia

(Rogers, 1994; Rose and Voynow, 2006; Voynow et al.,

2006), with goblet cell metaplasia and mucus hyperse-

cretion being stereotypical responses to the T cell

cytokines IL-4, IL-9 and IL-13 (Kim et al., 1997;

Dabbagh et al., 1999; Cohn et al., 2002, 2004; Zhou

et al., 2002; Reader et al., 2003). To our knowledge,

immune-mediated intestinal changes as they relate to

mucogenic responses to coccidial infection in the

chicken have not been defined previously.

In a mouse model, coccidial infection enhanced the

expression of IL-13 and subsequently IL-4, as well as

C.T. Collier et al. / Veterinary Immunology and Immunopathology 122 (2008) 104–115 113T

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the inflammatory cytokine IFN-g (Smith and Hayday,

2000). The present data demonstrate that mucin gene

expression was elevated above the non-infected base-

line in all infected birds at day 20 and further increased

at day 22 in C. perfringens-infected birds (without

narasin), while IL-4 expression was not significantly

elevated until day 22. Similarly, crypt goblet cell

numbers and villus goblet cell theca area were greatest

in birds infected with C. perfringens (alone and with

Eimeria) while crypt goblet cell theca area was largest

in birds infected only with coccidia. While these data

are consistent with previous reports demonstrating

mucogenic responses to IL-4 (Dabbagh et al., 1999), the

results further indicate that cytokines in addition to IL-4

or other bioactive factors must account for the enhanced

mucin expression. Possibilities would include IL-9 and

IL-13, which are well-documented modulators of goblet

cell functions (Louahed et al., 2000; Brown et al., 2001;

Cohn et al., 2002, 2004).

At the early phase of disease (day 20), IL-10 and

IFN-g, but not IL-4 expression were elevated only in

birds co-infected with Eimeria and C. perfringens. At

the peak of infection at day 22, the expression of these

cytokines continued to be greatest in co-infected birds.

On the other hand, IL-10 was elevated and IFN-g

suppressed in birds infected only with Eimeria at peak

infection. Coccidial-induced IL-10 expression has been

implicated as a strategy used by the parasite to suppress

inflammation and evade macrophage-mediated immune

destruction (Gazzinelli et al., 1992). In chicks, Rothwell

et al. (2004) observed enhanced expression of IFN-g in

the intestine following E. maxima infection. Further-

more, in a comparison of inbred lines of chickens that

differ in their resistance or susceptibility to Eimeria

infection, those investigators observed increased intest-

inal IL-10 mRNA expression in susceptible but not

resistant birds after E. maxima infection, while IFN-g

mRNA expression was similarly increased in both lines

of birds (Rothwell et al., 2004). Because their

observations were made at time points earlier after

infection than those in the present study, it is difficult to

compare the two studies. However, enhanced expres-

sion of IL-10 in birds infected only with Eimeria is

consistent with its known role as an immune

suppressive cytokine and in this case possibly provided

a mechanism for coccidia to evade immune-mediated

destruction.

The ionophore narasin significantly reduced cocci-

dial lesions in Eimeria-infected birds. This outcome

was associated with reduced goblet cell theca size in

villi, IL-10 and IFN-g expression on both days 20 and

22, the growth of mucolytic bacteria including C.

C.T. Collier et al. / Veterinary Immunology and Immunopathology 122 (2008) 104–115114

perfringens, coccidial and NE lesions and mortality in

birds that were co-infected with coccidia and C.

perfringens. Together these data are consistent with

the possibility that coccidiosis predisposes to the onset

of NE via mucogenesis. However, narasin can also

directly inhibit C. perfringens growth in vitro (Watkins

et al., 1997) and the possibility that it exerted a similar

effect in vivo was not tested in this study.

To our knowledge, the present data are among the

first to relate specific inflammatory responses to indices

of mucogenesis in the chicken intestine. Intriguingly,

both the chick NE and TPN piglet models (Ganessunker

et al., 1999; Craven et al., 2001; Collier et al., 2003)

demonstrate that inflammatory-mediated mucogenesis

in small intestinal mucosa, while being appropriate for

restoring barrier function, may indirectly lead to

secondary complications associated with bacterial

mucolysis. Therefore, the study also indicates the

importance of understanding the molecular basis of host

mucogenic responses to inflammatory cues and

determining the extent to which they impact the normal

microbiota. Such knowledge may have important

implications for a wide variety of chronic intestinal

inflammatory disorders of both animals and humans,

given that mucogenesis appears to be a stereotypical

response to local inflammation.

Acknowledgements

We thank Madelyn Stumpf and Jennifer Croix for

assistance with intestinal goblet cell histology and

Shankar Chowdhury, John Conour, and Noriko Naka-

mura for review of the manuscript. This study was

supported by Elanco Animal Health, Greenfield,

Indiana.

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