necrotic enteritis-producing strains of clostridium perfringens displace non-necrotic enteritis...

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Short communication Necrotic enteritis-producing strains of Clostridium perfringens displace non-necrotic enteritis strains from the gut of chicks Angelique J. Barbara, Hien T. Trinh, Robert D. Glock, J. Glenn Songer * Department of Veterinary Science and Microbiology, The University of Arizona, Tucson, AZ 85721, USA Received 17 May 2007; received in revised form 17 July 2007; accepted 20 July 2007 Abstract We inoculated broiler chicks with mixtures of Clostridium perfringens strains to investigate the single strain dominance observed in natural cases of necrotic enteritis (NE) [Nauerby, B., Pedersen, K., Madsen, M., 2003. Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens. Vet. Microbiol. 94, 257–266]. Pre-inoculation bacteriologic culture of chick intestines yielded up to six pulsed-field gel electrophoresis (PFGE) types of C. perfringens. Birds developed typical NE lesions in response to administration (2 per day for 4 days) of a combined inoculum comprising one NE strain (JGS4143, PFGE pattern 8) and four non-NE strains (from piglet necrotizing enteritis, chicken normal flora, human gas gangrene, and bovine neonatal enteritis). After inoculation commenced, only the NE strain was recovered through the first post-inoculation day, in spite of intense efforts to recover pre-challenge flora strains and the other challenge strains. Thereafter, pre-inoculation and previously undetected PFGE types were found, and JGS4143 became undetectable. Birds inoculated simultaneously with five NE strains (from disease in chickens or turkeys, and including JGS4143) also developed lesions, but again only JGS4143 was recovered through the 1st day post-challenge. At that time, birds began to be repopulated with pre-challenge PFGE types. Two NE strains (JGS4143 and JGS4064) produced bacteriocins, which inhibited each other and normal flora strains (n = 17), while normal flora strains inhibited neither NE strains nor each other. Thus, it appears that naturally occurring dominance of the gut by NE strains can be reproduced experimentally. Bacteriocins directed against normal flora could possibly provide the necessary advantage, although inhibition of one NE strain by another suggests that other factors may be partially or completely responsible for the dominance. # 2007 Elsevier B.V. All rights reserved. Keywords: Poultry necrotic enteritis; Clostridium perfringens; Bacteriocin 1. Introduction Poultry necrotic enteritis (NE) is commonly caused by Clostridium perfringens type A (Parish, 1961; Elwinger et al., 1992; Songer, 1996; Williams, 2005) and occurs worldwide (Songer, 1996; Williams et al., 2003). C. perfringens type A is a member of the www.elsevier.com/locate/vetmic Veterinary Microbiology 126 (2008) 377–382 * Corresponding author. Tel.: +1 520 621 2962; fax: +1 520 621 6366. E-mail address: [email protected] (J. Glenn Songer). 0378-1135/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2007.07.019

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Page 1: Necrotic enteritis-producing strains of Clostridium perfringens displace non-necrotic enteritis strains from the gut of chicks

www.elsevier.com/locate/vetmic

Veterinary Microbiology 126 (2008) 377–382

Short communication

Necrotic enteritis-producing strains of Clostridium perfringens

displace non-necrotic enteritis strains from the gut of chicks

Angelique J. Barbara, Hien T. Trinh, Robert D. Glock, J. Glenn Songer *

Department of Veterinary Science and Microbiology, The University of Arizona, Tucson, AZ 85721, USA

Received 17 May 2007; received in revised form 17 July 2007; accepted 20 July 2007

Abstract

We inoculated broiler chicks with mixtures of Clostridium perfringens strains to investigate the single strain dominance

observed in natural cases of necrotic enteritis (NE) [Nauerby, B., Pedersen, K., Madsen, M., 2003. Analysis by pulsed-field gel

electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens. Vet. Microbiol. 94, 257–266].

Pre-inoculation bacteriologic culture of chick intestines yielded up to six pulsed-field gel electrophoresis (PFGE) types of C.

perfringens. Birds developed typical NE lesions in response to administration (2� per day for 4 days) of a combined inoculum

comprising one NE strain (JGS4143, PFGE pattern 8) and four non-NE strains (from piglet necrotizing enteritis, chicken normal

flora, human gas gangrene, and bovine neonatal enteritis). After inoculation commenced, only the NE strain was recovered

through the first post-inoculation day, in spite of intense efforts to recover pre-challenge flora strains and the other challenge

strains. Thereafter, pre-inoculation and previously undetected PFGE types were found, and JGS4143 became undetectable.

Birds inoculated simultaneously with five NE strains (from disease in chickens or turkeys, and including JGS4143) also

developed lesions, but again only JGS4143 was recovered through the 1st day post-challenge. At that time, birds began to be

repopulated with pre-challenge PFGE types. Two NE strains (JGS4143 and JGS4064) produced bacteriocins, which inhibited

each other and normal flora strains (n = 17), while normal flora strains inhibited neither NE strains nor each other. Thus, it

appears that naturally occurring dominance of the gut by NE strains can be reproduced experimentally. Bacteriocins directed

against normal flora could possibly provide the necessary advantage, although inhibition of one NE strain by another suggests

that other factors may be partially or completely responsible for the dominance.

# 2007 Elsevier B.V. All rights reserved.

Keywords: Poultry necrotic enteritis; Clostridium perfringens; Bacteriocin

* Corresponding author. Tel.: +1 520 621 2962;

fax: +1 520 621 6366.

E-mail address: [email protected] (J. Glenn Songer).

0378-1135/$ – see front matter # 2007 Elsevier B.V. All rights reserved

doi:10.1016/j.vetmic.2007.07.019

1. Introduction

Poultry necrotic enteritis (NE) is commonly caused

by Clostridium perfringens type A (Parish, 1961;

Elwinger et al., 1992; Songer, 1996; Williams, 2005)

and occurs worldwide (Songer, 1996; Williams et al.,

2003). C. perfringens type A is a member of the

.

Page 2: Necrotic enteritis-producing strains of Clostridium perfringens displace non-necrotic enteritis strains from the gut of chicks

A.J. Barbara et al. / Veterinary Microbiology 126 (2008) 377–382378

intestinal flora of birds, but normal flora strains do not

produce lesions following experimental inoculation.

Strains from cases of NE are virulent, producing

disease of greater-or-lesser severity in experimentally

inoculated chicks (unpublished data).

C. perfringens is genetically diverse (Myers et al.,

2006), but isolates from birds in an NE outbreak are

usually genetically identical at the discriminatory

level of pulsed-field gel electrophoresis (PFGE). Up to

five PFGE types were isolated from normal birds in

Danish broiler flocks (Nauerby et al., 2003), but

examination of isolates from NE-affected chickens

revealed a single PFGE type in each flock. PFGE types

detected during NE outbreaks were not detected

before or after the outbreak, suggesting that, under

appropriate (but unknown) conditions, NE strains have

a competitive advantage over normal flora.

Elucidation of mechanisms by which NE strains

displace non-NE strains from the chicken intestine

may, in time, reveal information about pathogenesis

and provide targets for management of NE. We

present here the results of experimental coinfection

studies, the findings of which parallel those in natural

outbreaks and demonstrate that NE strains displace

non-NE strains from the gut of affected chicks.

2. Materials and methods

2.1. Bacteria and cultivation

Strains of C. perfringens used in these studies

(Table 1) were cultivated routinely on brain heart

infusion (BHI) agar (Difco, Detroit, MI) with 5%

citrated bovine blood. Plates were incubated at 37 8C

Table 1

Clostridium perfringens strains used in these studies

Strain number Study Source

JGS1235 2 Chicken

JGS1473 1 Chicken

JGS1521 2 Chicken

JGS1882 1 Porcine N

JGS1936 1 Bovine n

JGS4064 2 Chicken

JGS4104 2 Turkey N

JGS4143 1/2 Chicken

JGS4151 1 Strain 13

None N/A Normal fl

in an atmosphere of 5% H2:5% CO2:90% N2. Storage

of strains was in 50% glycerol at �80 8C.

2.2. Experimental reproduction of NE

Day-old, female Jumbo Cornish � Rock broiler

chicks (n � 25 per group; McMurray Hatchery,

Webster City, IA), housed in one-third of 1.5 m

circular brooders, were fed a commercial chick starter

on days 1–7 and a 28% protein wheat-based ration

mixed 1:1 with fishmeal thereafter. Water was

available ad libitum. Bedding (fine wood shavings)

was replaced at 2-week intervals. Environmental

temperature was maintained by allowing birds to self-

regulate their proximity to infrared heat lamps. On day

14, food was withheld for 20 h, followed by challenge.

Challenge inoculum was prepared by serial passage

through increasing volumes of cooked meat medium

(CMM; Difco) and fluid thioglycollate medium (FTG;

Difco), to a final FTG culture volume of 1 L.

Individual challenge inocula were administered alone

or combined to provide equal numbers of colony-

forming units of each challenge strain. In either case,

inocula were mixed 1.25:1 with high protein feed and

offered to chicks ad libitum, twice daily for 4 days.

Based upon loss of an estimated 25% of feed:inoculum

mixture due to scratching behavior and the routine

consumption of all remaining mixture, we estimate

that each chick consumed �3 � 109 colony-forming

units at each of the eight feedings.

2.3. Individual chick inoculation studies

In Study 1, the principal group was inoculated with

one NE strain (JGS4143) and four non-NE strains

Genotype

NE, hepatitis A, cpb2 pos

normal flora A, cpb2 pos

NE A, cpb2 pos

E case A, cpb2 pos

eonatal enteritis case A, cpb2 neg

NE A, cpb2 neg

E A, cpb2 pos

NE A, cpb2 pos

(human gas gangrene) A, cpb2 pos

ora from chicks in these studies A, cpb2 neg

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A.J. Barbara et al. / Veterinary Microbiology 126 (2008) 377–382 379

(JGS1882, JGS1473, JGS4151, and JGS1936). In

Study 2, principal group chicks were challenged with

five NE strains (JGS4143, JGS1235, JGS1521,

JGS4104, and JGS4064). Relative virulence of NE

strains (based upon percent of birds developing lesions

after inoculation) was 4143 > 4104 > 1521 >4064 > 1235. In both studies, positive control chicks

were challenged with JGS4143 and negative controls

were offered feed mixed with uninoculated FTG.

2.4. Bacteriologic and pathologic examination of

inoculated chicks

Two to four chicks were euthanized by CO2

asphyxiation, at intervals beginning 5 days before

challenge and continuing through 5 days post-

challenge. Intestines were removed and opened

aseptically, and jejunum and ileum were examined

for gross lesions. Segments (�10 mm) were fixed in

10% phosphate-buffered formalin and paraffin-

embedded. Five micrometre sections were stained

with haemotoxylin and eosin and examined for

microscopic lesions. To maximize the rigor of

attempts to recover PFGE types representative of

the population at the time of necropsy, the entire

mucosal layer was removed from a 10 cm segment of

jejunum and ileum by scraping with sterile micro-

scope slides. This material was examined by

bacteriologic culture on SPS agar (Merck, Rahway,

NJ), with results expressed as 1+ (growth in the initial

streak only) to 4+ (growth in the initial area of the

streak, plus the three parts of the streak for isolation).

All isolated colonies were collected for examination

by PFGE and for genotyping by a multiplex PCR

method (Meer and Songer, 1997).

2.5. PFGE typing of isolates

Each isolate or strain was cultivated overnight in

10 ml NIH thioglycollate broth with 1% starch and

180 mg chloramphenicol per ml. Cells harvested by

centrifugation (4000 � g, 20 min) were washed once

with SE wash buffer (75 mM NaCl, 25 mM EDTA (pH

8)) and resuspended to an OD600 of 1.3 in cell

suspension buffer (10 mM Tris (pH 7.2), 20 mM

NaCl, 50 mM EDTA). Cells from 1 ml of this

suspension were harvested by centrifugation and

resuspended in 50 ml of the same buffer. The

suspension was equilibrated to 56 8C, combined with

an equal volume of 2% CleanCut agarose (BioRad,

Hercules, CA), mixed gently, and poured into plug

molds. A 1 ml volume of plugs was suspended in 5 ml

of lysozyme buffer (10 mM Tris (pH 7.2), 50 mM

NaCl, 0.2% sodium deoxycholate, 0.5% sodium lauryl

sarcosine, and 1 mg lysozyme per ml) and incubated

for 16 h at 37 8C. Buffer was then removed and plugs

rinsed with proteinase K buffer (100 mM EDTA (pH

8.0), 0.2% sodium deoxycholate, and 1% sodium

lauryl sarcosine). Proteinase K reaction buffer

(100 mM EDTA (pH 8.0), 0.2% sodium deoxycholate,

1% sodium lauryl sarcosine, and proteinase K (1 mg

per ml)) was added (5 ml buffer for each ml of plugs),

followed by overnight incubation at 56 8C without

agitation. Plugs were then washed four times with

50 ml wash buffer (20 mM Tris (pH 8.0), 50 mM

EDTA) and stored at 4 8C.

Plugs suspended in 200 ml of NE buffer 4 (New

England Biolabs, Ipswich, MA) were incubated at

25 8C for 15 min and then 12 U of SmaI (New

England Biolabs) were added, followed by overnight

incubation at 25 8C. Buffer was then removed and

plugs incubated in 1 ml of 0.66� TBE buffer (60 mM

Tris–60 mM boric acid–1.3 mM EDTA (pH 8.3)) for

�30 min with gentle agitation. Plugs were cut to size

with a razor blade and each was placed on a tooth of

the comb. One percent low-melt preparative-grade

agarose (BioRad) was cast around the comb, which

was then removed, leaving the plug in place. PFGE

was performed in a CHEF-DR II apparatus (BioRad)

with pulse times ramped from 0.5 to 40 s over 20 h.

Gels were photographed via UV transillumination and

banding patterns were analyzed visually. PFGE

patterns were given arbitrary numbers.

2.6. Bacteriocin production

Each strain was cultivated to late log phase in BHI,

as above. An aliquot was adjusted to a density

equivalent to McFarland No. 0.5, and 100 ml amounts

were spread on BHI agar plates to prepare lawns. A

colony of each bacteriocin test strain was collected on

the tip of a sterile toothpick and stabbed through the

lawn and partially through the agar beneath. Plates

were incubated for 24 h at 37 8C under anaerobic

conditions and diameters of zones of inhibition were

measured in mm.

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A.J. Barbara et al. / Veterinary Microbiology 126 (2008) 377–382380

2.7. Approval of animal use

Study protocols were reviewed and approved a

priori by the University of Arizona Animal Care and

Use Committee.

3. Results

3.1. Genetic characterization of C. perfringens

recovered pre-, during, and post-challenge

Study 1 birds necropsied up to and including the

first inoculation day had no gross or microscopic

lesions. Isolates from these birds comprised six PFGE

types, all distinct from those of the inoculum strains.

Mild hyperemia of jejunal mucosa was observed on

the second inoculation day, without other lesions, but

the striking change was the disappearance of strains

with pre-challenge PFGE types (Table 2). Lesions of

varying severity but typical of NE appeared on day 3

of inoculation and were present through the first day

post-inoculation. In spite of rigorous attempts to

Table 2

Bacteriologic and pathologic examination of Study 1 birds, before, durin

Time Group

Pre-challenge N/A

During challengea Positive control

During challengeb Negative control

During challengea Five strains

Post-challengec Five strains, positive control

a Collected on challenge days 1–3.b Collected on challenge day 3.c Collected on post-challenge days 1–3.

Table 3

Bacteriologic and pathologic examination of Study 2 birds, before, durin

Time Group

Pre-challenge N/A

During challengea Positive control

During challengeb Negative control

During challengea Five strains

Post-challengec Positive control

Post-challengec Five strains

a Collected on challenge days 1–3.b Collected on challenge day 3.c Collected on post-challenge days 1–3.

isolate any strain that was present, only PFGE type 8

(inoculum strain JGS4143) was recovered through the

first post-inoculation day. After the first post-inocula-

tion day, pre-inoculation PFGE types were again

found, accompanied by PFGE type 8 and previously

undetected type 16. Thereafter, PFGE type 8 was not

isolated again, and types 17 and 18 appeared.

Study 2 birds yielded five PFGE types pre-challenge,

none of which matched the patterns of the inoculum

strains (Table 3). The pattern of lesion development and

resolution was the same as in Study 1. During the 4 days

of challenge, PFGE type 8 was isolated from all birds

challenged with five NE strains, and PFGE type 10 (not

an inoculum type) was also found in a few. Strains

JGS1235, JGS1521, JGS4064, and JGS4104 had each

produced signs and lesions of NE in experimentally

inoculated birds (unpublished data). However, they

were not recovered from birds inoculated with these 4 in

the company of JGS4143 (PFGE type 8) in this study.

PFGE type 8 alone was found in positive control birds

during challenge, and PFGE types 8, 10, 12, 15, 16, and

17 were found post-challenge in positive controls and in

birds challenged with the mix of five strains.

g, and after inoculation

Average lesion score PFGE strain number(s)

0 10, 11, 12, 13, 14, 15

2.3 8

0 10, 15

2.4 8

0 8, 12, 13, 14, 15, 16, 17, 18

g, and after inoculation

Average lesion score PFGE strain number(s)

0 10, 11, 12, 13, 14

2.4 8

0 15

2.2 10, 8

0.9 10, 8, 16

1.0 8, 12, 15, 17

Page 5: Necrotic enteritis-producing strains of Clostridium perfringens displace non-necrotic enteritis strains from the gut of chicks

A.J. Barbara et al. / Veterinary Microbiology 126 (2008) 377–382 381

Table 4

Bacteriocin production (zone diameters in mm) of NE and non-NE strains

Bacteriocin test strains

4064 4143

Lawns 4064 0 5.6a

4143 5.7 0

Normal flora strains (n = 17) 6.6b (range 3.5–11)c 6.8 (range 4–11)

A zone denoted ‘‘0’’ indicate no inhibition.a Average of three repetitions.b Average across all 17 normal flora strains.c Range of averages (each based upon three repetitions) for normal flora strains.

3.2. Bacteriocin production by NE and non-NE

strains

All NE strains inhibited growth of normal flora, but

normal flora strains did not inhibit any NE strain (i.e.,

there was no zone of inhibition). Representative data

are presented in Table 4. Two NE strains (JGS4143

and JGS4064) inhibited each other and normal flora

strains (n = 17). Normal flora strains did not inhibit

each other (data not shown).

4. Discussion

Replacement of the genetically heterogeneous

(based upon PFGE analysis) enteric population of

C. perfringens, usually by single, chicken-virulent

strains has been noted in natural outbreaks of NE

(Engstrom et al., 2003; Nauerby et al., 2003). We

reasoned that experimental reproduction of naturally

occurring strain dominance would facilitate study of

population dynamics of C. perfringens in infected

chicks and perhaps lead to identification of specific

virulence attributes. Genome hyperplasticity (Myers

et al., 2006) augurs against use of PFGE to draw

definitive conclusions about relatedness among

diverse strains, but disparate PFGE types imply

variety in the population; strains with identical PFGE

types are likely to be identical, especially when

comparing inoculum and output strains from the same

birds: it is rational to assume that appearance of an

inoculum PFGE type in post-inoculation cultures is

the result of inoculation. Thus, PFGE was useful in

these studies for tracking both normal flora and

challenge strains. The disappearance of pre-challenge

PFGE types upon challenge with NE strains and the

disappearance of PFGE type 8 (NE strain JGS4143)

and reappearance of pre-inoculation and new PFGE

types upon recovery has the appearance of the sort of

strain dominance seen in the field (Engstrom et al.,

2003; Nauerby et al., 2003). Apparent inhibition of

other NE strains by JGS4143 suggests that there is no

equality in this group.

It is possible that the pre- and post-inoculation

strains were in the gut throughout the study and simply

went undetected due to small numbers. Our data do

not rule out this possibility, but it seems no more likely

than that birds became re-colonized through exposure

to their previous flora strains in the environment. In

any case, the point is the same: strains which were

readily detectable, or introduced via challenge, were

greatly reduced in numbers or eliminated in the

presence of an NE strain.

These experimental results confirm those of

individual inoculation studies that differences exist

in virulence for chicks among C. perfringens strains. It

seems clear that NE strains have a selective advantage

over non-NE strains and even degrees of selective

advantage over each other. The last finding suggests

that variations in strain virulence may express

themselves not only in the proportion of birds

developing lesions upon challenge, but also in

interstrain competition for a place in the intestine.

These findings are in keeping with those obtained

from studying natural disease (Engstrom et al., 2003;

Nauerby et al., 2003).

There is no evidence that differences in generation

time vary significantly among NE strains or between

NE and non-NE strains (unpublished data). This

should perhaps be explored further. However, bacter-

iocin action is a documented means by which bacteria

compete for resources and C. perfringens bacteriocin

Page 6: Necrotic enteritis-producing strains of Clostridium perfringens displace non-necrotic enteritis strains from the gut of chicks

A.J. Barbara et al. / Veterinary Microbiology 126 (2008) 377–382382

production has been reported (Dupuy et al., 2005;

Dupuy and Matamouros, 2006). All NE strains

examined in this study produced substances that

strongly inhibited non-NE strains; the reverse did not

occur, nor did non-NE strains inhibit each other. As far

as we are aware, there is no information on in vivo

bacteriocin production by C. perfringens, but it is

tempting to speculate that elaboration of bacteriocins in

the gut allows displacement of normal flora strains and

anarchic multiplication by NE strains. This is tempered

by the fact that strains JGS4143 and JGS4064 inhibited

each other about equally in vitro and JGS4143 out-

competed JGS4064 in vivo. Thus, the phenomenon may

be due, completely or in part, to factors other than

bacteriocins, such as superior adhesion characteristics,

more rapid multiplication, and production of specific

toxins. Further study is indicated.

Acknowledgements

The authors acknowledge, with gratitude, the

technical assistance of Jeremy Coombs. Supported in

part by a grant from Schering-Plough Animal Health.

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