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Veterinary Microbiology 157 (2012) 412–419

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he effect of Clostridium perfringens type C strain CN3685 and its isogeniceta toxin null mutant in goats

. Garcia a, J. Beingesser a, D.J. Fisher b,c,1, S. Sayeed b,2, B.A. McClane b,c,. Posthaus d, F.A. Uzal a,*

alifornia Animal Health and Food Safety Laboratory System, San Bernardino Branch, School of Veterinary Medicine,

iversity of California, Davis, San Bernardino, CA 92408, USA

epartment of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

olecular Virology and Microbiology Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

stitute of Animal Pathology, Vetsuisse Faculty, University of Bern, PO-box 8466, 3001 Bern, Switzerland

1. Introduction

Clostridium perfringens is an anaerobic, spore-forming,Gram-positive rod that is a pathogen of humans, domesticand wild animals (Miclard et al., 2009). C. perfringens

produces a vast array of toxins, including four toxins thatare used to classify C. perfringens isolates into five types (A–E); depending upon production of these four typing toxins,namely (alpha [CPA], beta [CPB], epsilon [ETX] and iota[ITX]) (Uzal and McClane, 2011). All types produce CPA; in

R T I C L E I N F O

icle history:

ceived 31 August 2011

ceived in revised form 31 December 2011

cepted 4 January 2012

ywords:

ta toxin

stridium perfringens type C

terotoxemia

ats

A B S T R A C T

Clostridium perfringens type C is an important cause of enteritis and/or enterocolitis in

several animal species, including pigs, sheep, goats, horses and humans. The disease is a

classic enterotoxemia and the enteric lesions and associated systemic effects are thought

to be caused primarily by beta toxin (CPB), one of two typing toxins produced by C.

perfringens type C. This has been demonstrated recently by fulfilling molecular Koch’s

postulates in rabbits and mice. We present here an experimental study to fulfill these

postulates in goats, a natural host of C. perfringens type C disease. Nine healthy male or

female Anglo Nubian goat kids were inoculated with the virulent C. perfringens type C wild-

type strain CN3685, an isogenic CPB null mutant or a strain where the cpb null mutation

had been reversed. Three goats inoculated with the wild-type strain presented abdominal

pain, hemorrhagic diarrhea, necrotizing enterocolitis, pulmonary edema, hydropericar-

dium and death within 24 h of inoculation. Two goats inoculated with the CPB null mutant

and two goats inoculated with sterile culture media (negative controls) remained

clinically healthy during 24 h after inoculation and no gross or histological abnormalities

were observed in the tissues of any of them. Reversal of the null mutation to partially

restore CPB production also increased virulence; 2 goats inoculated with this reversed

mutant presented clinical and pathological changes similar to those observed in goats

inoculated with the wild-type strain, except that spontaneous death was not observed.

These results indicate that CPB is required for C. perfringens type C to induce disease in

goats, supporting a key role for this toxin in natural C. perfringens type C disease

pathogenesis.

� 2012 Elsevier B.V. All rights reserved.

Corresponding author at: California Animal Health and Food Safety

oratory-San Bernardino Branch, University of California-Davis, 105

est Central Avenue, San Bernardino, CA 92408, USA.

l.: +1 909 383 4287; fax: +1 909 884 5980.

E-mail address: fuzal@cahfs.ucdavis.edu (F.A. Uzal).

Department of Microbiology and Immunology, Uniformed Services

iversity of the Health Sciences, Bethesda, MD, USA.

Department of Environmental and Occupational Health, Graduate

ool of Public Health University of Pittsburgh, PA 15261, USA.

Contents lists available at SciVerse ScienceDirect

Veterinary Microbiology

jou r nal h o mep ag e: w ww .e ls evier . co m/lo c ate /vetm i c

78-1135/$ – see front matter � 2012 Elsevier B.V. All rights reserved.

i:10.1016/j.vetmic.2012.01.005

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J.P. Garcia et al. / Veterinary Microbiology 157 (2012) 412–419 413

ddition, C. perfringens type B and C isolates produce CPB,pe B and D isolates produce ETX and type E isolates

roduce ITX.C. perfringens type C causes highly lethal diseases in

any mammalian species that are characterized mainly byecrotizing enteritis or enterocolitis. Most clinical signsnd lesions associated with C. perfringens type C disease areought to be produced by CPB, a 35-kDa protein thatrms pores in the membrane of susceptible cell lines,ading to cell swelling and lysis (Uzal and McClane, 2011).PB is destroyed by proteolytic enzymes such as trypsin,xplaining why the neonate is especially predisposed toPB action; i.e., the trypsin inhibitor action of colostrum,hich prevents proteolytic degradation of immunoglobu-

ns during the first days of life, also protects CPB (Diabt al., 2011; Smith and Sherman, 1996).

Death is likely to result primarily from toxemiallowing absorption of CPB from the intestine into

irculation, allowing targeting of internal organs (Mat al., 2011; Uzal et al., 2009). It is also possible that otherxins present in the intestinal lumen gain access to the

lood circulation following massive intestinal mucosalarrier damage, and that they contribute to the systemicffects observed in C. perfringens type C disease.

The lethal and necrotizing effects of CPB play keyoles in the tissue damage that is characteristic of C.erfringens type C infections (McClane et al., 2004). Thisas demonstrated when we recently constructed a

eries of C. perfringens type C toxin null mutants in strainN3685, which demonstrated that CPB, but not CPA orerfringolysin O (PFO), is necessary for type C isolates toause intestinal damage in a rabbit intestinal loop modelnd lethality in mice (Sayeed et al., 2008; Uzal et al.,009).

In goats, information about the pathogenesis of C.

erfringens type C infection is scant. To gain knowledge one mechanism of type C infection in this species, we

eveloped a model for type C infection in goats, and thensted the CPB null mutant and a reversed mutant strain inis model, fulfilling molecular Koch’s postulates in a

atural host of C. perfringens type C disease.

. Materials and methods

.1. Animals

Conventionally reared, healthy male and femalenglo Nubian goat kids were used. They were neveraccinated against clostridial or other disease and hadeen weaned at the age of 10–14 weeks (an age at whichost maternal antibodies have disappeared), placed in aarm, dry room, and fed alfalfa hay and water ad libitum

r 1 or 2 weeks before the experiments. All animalsere between 14 and 20 kg body weight. Fecal samplesere collected 4 and 2 days before the beginning of the

xperiments. A fecal float analysis to detect intestinalarasite eggs and parasites was performed with each ofhese samples. All procedures involving animals wereeviewed and approved by the University of California,avis Committee for Animal Care and Use (permit6383).

2.2. Bacterial strains

C. perfringens type C isolate CN3685 was provided byRussell Wilkinson (University of Melbourne, Australia).This isolate, originally from the Burroughs-Welcome C.

perfringens collection, had been isolated in 1954 fromperitoneal fluid of a sheep with struck. It has beenpreviously characterized (Fisher et al., 2006) for itsgenotype and toxin production, which showed that (underthe growth conditions used in our current study) thisisolate produces �13 mg/ml of CPB. This toxin productionlevel places CN3685 among the strongest 20% of CPBproducers of the 45 C. perfringens type C disease isolatessurveyed in that previous study (Fisher et al., 2006).Isogenic CN3685 CPB toxin null mutant BMC 104 had beenconstructed previously by insertion of a targeted group IIintron into the cpb gene; characterization studies con-firmed that this insertional event had inactivated CPBproduction (Sayeed et al., 2008). The reversed CPB mutantBMC 105 had also been constructed previously byintroduction of an Ltr-encoding plasmid into BMC104.This allows removal of the intron from some disrupted cpb

message, which is apparently transcribed as a monocis-tronic mRNA in BMC104, partially restoring CPB produc-tion by the reversed mutant to about one third of wild-typelevels (Sayeed et al., 2008).

2.3. Production of type C cultures for goat inoculation

Wild-type CN3685, BMC 104 and BMC 105 were eachgrown for 8 h in TGY (tryptone, yeast extract, glucose,cysteine hydrochloride and distilled water) at 37 8C. Thereversed CPB mutant BMC105 was grown in the presenceof 15 mg/ml of chloramphenicol to retain the LtrA-encoding plasmid needed to remove the intron insertionfrom the cpb message and partially restore CPB production.A 10% inoculum (50 ml) of each starter culture was thentransferred into 500 ml of fresh TGY and grown at 30 8Covernight (log phase culture); in BMC105, this incubationinduced the expression of the LtrA protein required fortrans-splicing removal of the sense oriented intron fromthe cpb mRNA (results not shown). Those culture super-natants were assayed by Western blotting (data notshown) as previously described (Sayeed et al., 2008), toconfirm CPB production by CN3685 and BMC105, but notBMC104.

2.4. Intraduodenal inoculation of C. perfringens type C strain

CN3685 and its derivatives in goats

The animals were divided into 4 treatment groups asfollows. Three animals were inoculated with the wild-typestrain CN3685. Two animals each were inoculated with theCPB null mutant BMC 104, the reversed CPB mutant BMC105 or sterile, non-toxic TGY (negative controls). The goatkids were fasted for 24 h before inoculation but wereallowed access to water until 1 h before surgery. The pre-anesthetic induction was carried out by intravenousinjection of a mixture of ketamine and diazepam, andanesthesia was maintained with a mixture of isofluoraneand oxygen administered by endotracheal tube in an open

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J.P. Garcia et al. / Veterinary Microbiology 157 (2012) 412–419414

cuit. A laparotomy was performed via the right flank,d the pyloric area of the abomasum and the first portion

duodenum were exposed. Two hundred ml of a 20%lution of wheaten corn flour in 0.85% saline werejected into the abomasum of all animals in each group.en, 250 ml of a culture of the wild-type, CPB null mutant

reversed mutant were inoculated into the duodenumrough a drip, over a period of approximately 10 min.ntrol animals received 250 ml of sterile, non-toxic,rile TGY instead of bacterial culture. All animals

ceived 0.25 g of trypsin inhibitor (Sigma, St. Louis,issouri) mixed with the inoculum. The abdominalcision was closed by separate muscle and skin sutures.

The animals were allowed to recover from anesthesiad then clinically examined periodically after inocula-n. Those animals that remained clinically healthyring that period, and those that showed moderate or

ild clinical signs, were euthanized 24 h after inoculation.thanasia was performed by an intravenous overdose ofrbiturate.

. Clinical signs

A scoring system from 0 to 3 was used to characterizeerall clinical abnormalities. A score of 0 was assigned toimals that did not show clinical abnormalities during the

hole experimental period. A score of 1 was assigned toimals that showed only mild, transient diarrhea, andat reminded alive during this time. A score of 2 wassigned to animals that showed moderate, hemorrhagicarrhea accompanied by abdominal discomfort andpression during most of the observation period, andat reminded alive during this time. A score of 3 wassigned to animals that showed severe hemorrhagicarrhea and abdominal pain and depression, dyingontaneously within 24 h after inoculation.

. Post-mortem examination, histopathology, microbiology

sting and ELISA for C. perfringens toxins

Post-mortem examinations were performed immedi-ely after death on all goats. Samples of the gastro-testinal tract, brain, kidneys, lungs, heart, spleen ander were collected and fixed by immersion in 10%

rmalin pH 7.2 for a minimum of 24 h. After this, theains were sliced into sections 0.5 cm thick, and fixed insh formalin for 7 more days before blocks were preparedm corpus striatum, parietal cortex, midbrain at the level

anterior colliculi, pons, cerebellar peduncles, cerebellumd medulla at the level of the obex. All tissues wereocessed routinely to obtain 4 mm thick sections whichere stained with hematoxylin and eosin. Selectedctions were also stained with Gram or Phospho Tungsticid Hematoxylin (PTAH).Small and large intestinal contents of each goat and

mples of liver, spleen and lung were aseptically collectedd inoculated onto reduced Brucella Blood Agar (Anaerobicstems, Morgan Hill, California) and incubated anaerobi-lly or aerobically at 37 8C for 48 h. Anaerobic isolates wereentified by conventional biochemical techniques. C.

rfringens isolates were typed by a multiplex PCR technique

to amplify segments specific for the genes encoding CPA,CPB, ETX, ITX, CPE and CPB2, as previously described(Bueschel et al., 2003). Briefly, bacteria were grown onbrain-heart infusion agar plates (Hardy Diagnostics, SantaMaria, California), incubated anaerobically overnight at37 8C and then processed for multiplex PCR analysis usingcolony lysates as templates. The multiplex PCR productswere then separated in 2% agarose gels, stained withethidium bromide and examined by ultraviolet lighttransillumination.

Samples of small and large intestinal contents from allgoats were also tested for CPA, CPB and ETX via acommercial capture ELISA kit (BIO-X, Brussels, Belgium),following the manufacturer’s instructions. Briefly, the testused 96-flat-bottom-well plates sensitized by specificmonoclonal antibodies to CPA, CPB or ETX. Samples wereadded to wells and plates were incubated for 60 min atroom temperature, followed by washing and incubationfor 60 min with peroxidase-labeled anti-CPA, -CPB or -ETXpolyclonal antibodies. Plates were washed again, and amixture of chromogen-substrate (hydrogen peroxide andtetramethyl bendizine) was added. The enzymatic reactionwas stopped by acidification with phosphoric acid. Opticaldensities were read using an ELISA reader with a 450 nmfilter. Purified CPA, CPB or ETX were used in positivecontrol wells; toxins were replaced by buffer in negativecontrol wells. Results were calculated according to themanufacturer’s instructions.

2.7. Immunohistochemistry

All sections of small intestine and colon were processedby an indirect immunoperoxidase technique for C.perfringens, as previously described (Diab et al., 2011)using the Dako EnVision kit (Dako, Carpenteria, CA)according to the instructions of the manufacturer. A rabbitpolyclonal anti-C. perfringens antibody (GenWay Bio, SanDiego, CA) was used as primary antibody. Small intestinefrom a goat that was culture-negative for C. perfringens wasused as negative control. Also, sections of small intestineand colon from all goats were processed by an indirectimmunoperoxidase technique for CPB as previouslydescribed (Miclard et al., 2009) using the LSAB kit(DakoCytomation; Dako, San Diego, California) accordingto the instructions of the manufacturer. An anti-CPBmonoclonal antibody (Center for Veterinary Biologics,Ames, Iowa) was used as primary antibody.

3. Results

A summary of the overall severity of the clinical signs,gross and microscopic lesions is presented in Table 1. Allanimals inoculated with the wild-type isolate or with thereversed mutant presented hemorrhagic diarrhea,abdominal pain and depression, but these clinical signswere more severe in the 3 animals inoculated with thewild-type strain, which were the only animals that diedspontaneously. The animals inoculated with the CPB nullmutant and the negative controls did not show clinicalabnormalities, except for one of the goats inoculated withthe CPB null mutant strain that presented mild, transient

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J.P. Garcia et al. / Veterinary Microbiology 157 (2012) 412–419 415

iarrhea that resolved spontaneously 4 h after onset. Nother clinical abnormalities were observed in any othernimal.

A summary of the main gross changes is presented inable 2 and in Figs. 1–4. Gross lesions characterized mostlyy hemorrhagic fluid content with strands of fibrin,cipient pseudomembrane formation, mucosal hemor-

hage and congestion involving jejunum, ileum and spiralolon were observed only in animals inoculated with theild-type strain (Fig. 1). Two animals inoculated with theild-type also presented transmural edema of smalltestine and colon, and hemorrhagic mesenteric lymph

odes. Excess of hemorrhagic peritoneal and pleural fluidnd fibrin strands on the intestinal serosa were alsobserved in one of the animals inoculated with the wild-pe strain. The small intestine and colon of the 2 animalsoculated with the CPB reversed mutant strain presented

emorrhagic mucosa, fluid hemorrhagic content and fibrin the lumen (Fig. 3). The colon content of one animaloculated with the CPB null mutant was fluid. No other

ignificant gross abnormalities were observed in any of thenimals of the four groups.

A summary of the main microscopic findings in theintestinal tract is presented in Table 3 and Figs. 5–12.Microscopic lesions were observed only in animalsinoculated with the wild-type or CPB reversed mutant,but the severity of the lesions was greater in the animalsinoculated with the wild-type strain than in thoseinoculated with the CPB reversed mutant. When present,the lesions were similar in small intestine (affecting mostlyjejunum and ileum) and colon, but they were generallymore severe in the small intestine.

Most microscopic lesions were multifocal; however, thesubmucosa, muscularis and/or serosa, showed diffuseproteinaceous edema and mild inflammatory cell infiltra-tion in all the animals inoculated with the wild-type and inone of the goats inoculated with the CPB reversed mutant(Table 3). The small intestine showed severe villousblunting, coagulative mucosal necrosis and an inflamma-tory cell infiltrate composed of neutrophils and fewermacrophages, lymphocytes and plasma cells (Figs. 5, 7, 9and 11). Fibrin thrombi occluding superficial capillaries,arterioles or venules of the lamina propria were rarelyobserved. Numerous crypts were filled with neutrophils

able 1

ummary of overall severity of clinical signs, gross and microscopic lesions observed in 9 goats inoculated with C. perfringens type C wild-type, its mutants,

r with sterile non-toxic culture medium.

Inoculum Case No Interval

inoculation to

death

Death Overall

severity of

clinics signsa

Overall severity

of gross lesionsb

Overall severity

of microscopic

lesionsc

C. perfringens type C wild

type (CN3685)

1 22.5 h Spontaneous 3 2.5 3

2 6 h Spontaneous 3 3 3

3 3.20 h Spontaneous 3 3 3

C. perfringens type C CPB

null mutant (BMC 104)

4 24 h Euthanasia 1 0.5 0

5 24 h Euthanasia 0 0 0

C. perfringens type C CPB

reversed mutant (BMC 105)

6 24 h Euthanasia 2 1 2

7 24 h Euthanasia 2 1 2

Sterile, non-toxic TGY 8 24 h Euthanasia 0 0 0

9 24 h Euthanasia 0 0 0a 0: no clinical abnormalities; 1: mild, transient diarrhea; 2: moderate hemorrhagic diarrhea accompanied by abdominal discomfort and depression

uring most of the observation period; 3: similar signs to those with score 2, but of greater severity; animals died spontaneously within 24 h of inoculation.b 0: no gross lesions; 1–3: mild to severe gross lesions (see Table 2 for details).c 0: no microscopic lesions; 1–3: mild to severe microscopic lesions (see Table 3 for details).

able 2

ain gross lesions observed in 9 goats inoculated with C. perfringens type C wild-type, its mutants, or with sterile non-toxic culture medium.

Inoculum Case No Small intestinea Colona

Mucosal congestion

and hemorrhage

Fluid

content

Pseudomembrane Mucosal congestion

and hemorrhage

Fluid

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Pseudomembrane

C. perfringens type

C wild type (CN 3685)

1 3 3 2 0 3 2

2 3 2 3 0 3 3

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C. perfringens type C

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6 1 1 1 1 1 0

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8 0 0 0 0 0 0

9 0 0 0 0 0 0

a 0: no gross lesions; 1–3: mild to severe gross lesions.

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J.P. Garcia et al. / Veterinary Microbiology 157 (2012) 412–419416

d a moderate number of neutrophils were seenigrating through the crypt epithelium. The luminalrface of the intestine of two goats inoculated with theild-type was covered by a pseudomembrane composed

degenerated and necrotic desquamated epithelial cells,

cell debris, neutrophils, lymphocytes, plasma cells, macro-phages, fibrin and large thick bacilli with square ends andoccasional subterminal spores. Similar bacilli wereobserved, singly or in clusters, free in the intestinal lumen,or lining the margin of the denuded mucosal surface.

s. 1–12. (Fig. 1) Jejunum of a goat inoculated with C. perfringens type C wild-type. Observe the presence of a pseudomembrane and blood in the intestinal

en. (Fig. 2) Jejunum of a goat inoculated with C. perfringens type C CPB null mutant. No gross lesions are observed. (Fig. 3) Jejunum of a goat inoculated

th C. perfringens type C CPB reverse mutant. The mucosa is hemorrhagic and there is fibrin present in the intestinal lumen. (Fig. 4) Jejunum of a goat

culated with sterile, non-toxic TGY. No gross lesions are observed. (Fig. 5)Jejunum of a goat inoculated with C. perfringens type C wild type. The mucosa

d submucosa are diffusely necrotic. Bar: 100 mm. (Fig. 6) Jejunum of a goat inoculated with C. perfringens type C CPB null mutant. No significant

tological lesions are observed. Bar: 100 mm. (Fig. 7) Jejunum of a goat inoculated with C. perfringens type C CPB reverse mutant. There is diffuse mucosal

crosis. Bar: 100 mm. (Fig. 8) Jejunum of a goat inoculated with TGY. No significant histological lesions are observed. Bar: 100 mm. (Fig. 9) Higher

gnification of the goat’s jejunum showed in Fig. 5. There is complete loss of villus and crypt epithelium and necrosis of the lamina propria. Bar: 40 mm.

g. 10) Higher magnification of the goat’s jejunum showed in Fig. 6. No significant histological abnormalities are observed. Bar: 40 mm. (Fig. 11) Higher

gnification of the goat’s jejunum showed in Fig. 7. There is partial loss of villus epithelium. Bar: 40 mm. (Fig. 12) Higher magnification of the goat’s

unum showed in Fig. 8. No significant histological abnormalities are observed. Bar: 40 mm.

J.P. Garcia et al. / Veterinary Microbiology 157 (2012) 412–419 417

Although the bacterial population was usually greater inthe intestinal lumen, a few bacilli were also seen in cryptsand glands and invading the necrotic lamina propria. Morethan 95% of the bacilli observed in the lumen of the smallintestine and colon of the goats inoculated with any of theC. perfringens strains were Gram-positive and stainedstrongly for C. perfringens by immunohistochemistry. Thespiral colon crypts presented hypertrophy of the gobletcells and were full with abundant mucus.

Histological changes were not usually observed outsidethe gastrointestinal tract of any of these animals. However,moderate to severe kidney and liver congestion, and mildmyocardial hemorrhage developed in one animal inocu-lated with the reversed mutant. No significant histologicalabnormalities were observed in any tissues of the animalsinoculated with the CPB null mutant or TGY (Figs. 6, 8, 10and 12). Only a few thick Gram-positive bacilli with squareends were observed in the lumen of the small intestine andcolon of the animals inoculated with TGY; these bacillistained strongly positive by C. perfringens immunohisto-chemistry.

Only one animal inoculated with the wild-type strainshowed positive results after CPB immunohistochemistry.In this animal, positive staining of endothelial cells ofmedium size veins in the lamina propria and submucosawas observed. No CPB staining was observed in any of theintestinal sections of any other animal.

A small amount of mixed aerobic flora was isolatedfrom the small and large intestine of all goats. C. perfringens

was cultured from the small and large intestinal contentsof all the goats inoculated with the wild-type, CPB nullmutant and the reversed CPB mutant. The isolates fromwild-type and complemented strain-inoculated animalswere identified as type C by the toxin genotyping multiplexPCR assay. Those isolated from the animals inoculated withthe CPB null mutant were identified as type A by thismultiplex toxin genotyping PCR assay, which does notefficiently amplify a product from the intron-disrupted cpb

gene (data not shown). No other aerobic or anaerobicmicroorganisms were isolated from any of the othersamples from any of the goats cultured.

CPA and CPB were detected by ELISA in small and largeintestinal contents of all goats inoculated with the wild-type and the reversed mutant. CPA, but not CPB, wasdetected in the small and large intestinal content of thetwo animals inoculated with the CPB null mutant and thetwo animal inoculated with TGY.

4. Discussion and conclusions

The objective of this study was to comprehensivelydocument the lesions produced by C. perfringens type C ingoats and also to test the hypothesis that CPB is necessaryand sufficient to produce clinical signs and gross andmicroscopic lesions of C. perfringens type C disease in anatural host. In this study, the virulence of a wild-type C.

perfringens type C was completely eliminated when thegene encoding CPB was inactivated in a virulent wild-typeC strain. This virulence was partly restored when the CPBmutation was reversed in this isolate, thus indicating thatthe observed loss of pathogenicity for the CPB null mutantT

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J.P. Garcia et al. / Veterinary Microbiology 157 (2012) 412–419418

as not due to an unintended secondary mutation andlfilling molecular Koch postulates. Molecular Koch’sstulates for C. perfringens type C infection have beenlfilled before in rabbits and mice, where virulence wasminated when the gene that encodes CPB was inacti-ted in the virulent type C isolates and virulence wasstored by reversing this mutation (Sayeed et al., 2008).

It was notable that the animals inoculated with theild-type strain developed more severe clinical signs,oss and histopathological lesions than those inoculatedith the reversed mutant. This phenomenon was observedfore in rabbit intestinal loops and in mice inoculatedtraduodenally with this mutant and it is believed to be ansequence of the reversed mutant producing, both in

ro and in vivo, substantially less CPB than the wild-typecause of inefficient splicing to remove the intronsertion from the cpb mRNA (Sayeed et al., 2008).

The most consistent gross and histological finding inats inoculated with the wild-type strain in our study wascrotizing enterocolitis. This is similar to the lesionsscribed for spontaneous C. perfringens type C infections

other animal species, including horses, pigs and sheepiab et al., 2011; Songer and Uzal, 2005). Histologicidence of mucosal or submucosal thrombosis wasscribed before as a hallmark of type C infections inglets, horses and humans (Diab et al., 2011; Miclard et al.,09; Lawrence and Cooke, 1980). Miclard et al. (2009)ggested that mucosal thrombosis was an early event inpe C infections in piglets, leading to mucosal necrosis.wever, the study referred to Miclard et al. (2009)

vestigated end stage lesions of necrotizing enteritis and conclusions on initial stages of the disease could beawn. Our results lend support to the idea that, at least inats, CPB has a direct effect on the intestinal mucosa andat the mucosal necrosis observed in our goats was due toe primary action of CPB on the intestinal epitheliumther than ischemia secondary to vascular thrombosis. Ittherefore possible that the pathogenesis of C. perfringens

pe C disease is different in goats and in pigs. Never-eless, extensive hemorrhage and tissue edema wereesent in all animals inoculated with CPB-producingains, demonstrating that vascular leakage is an impor-

nt component of type C infection. CPB was demonstrated rapidly damage cultured endothelial cells (Gurtner et al.,10; Popescu et al., 2011) and was detected at vasculardothelial cells in one of the affected goats in our study.erefore it is possible that endothelial damage mediated

CPB also contributes to the pathogenesis of type Cteritis in goats.A presumptive diagnosis of C. perfringens type C disease

most animal species can be achieved by observingcrotizing enteritis or enterocolitis, followed by isolation

C. perfringens type C, since this type of C. perfringens isrely found in the intestine of healthy animals (Diab et al.,11). However, final confirmation of a type C infection

agnosis should be based on the detection of CPB intestinal contents (Diab et al., 2011). In this case, C.

rfringens type C was isolated from small and largetestinal content of all the animals inoculated with theild-type and the reversed mutant, and CPB was detected

confirming that the clinical signs, gross and histologicallesions observed were produced by C. perfringens type C.

No CPB was detected in the intestinal samples of theanimals inoculated with the CPB null mutant strain,confirming that this strain did not produce CPB. The C.

perfringens isolates from the intestine of the animalsinoculated with the CPB null mutant were typed by PCRas type A. This is due to the presence of an intron insertioninto the cpb gene in this strain, which (using cpb primers)would produce a PCR product that is poorly amplified understandard multiplex toxin genotyping PCR conditions.

A relatively small number of animals were used for theseexperiments due to humane reasons and also to reduce costs.It had initially been decided that the number of animals pergroup would be increased if needed after analyzing theresults of the first set of experiments. However, given thehigh degree of consistency of the results obtained, it wasdecided not to increase the number of animals.

None of the animals inoculated with the CPB nullmutant or TGY developed significant clinical signs, gross orhistological lesions. This confirms that the alterationsobserved in the animals inoculated with the wild-type orthe complemented strains were indeed due to CPB. Thisstudy therefore confirms that CPB is required to producethe clinical signs, gross and histological signs of C.

perfringens type C infection in goats.

Conflict of interest statement

The authors express that there are no conflicts of interestthat could bias the work presented in this manuscript.

Acknowledgments

National Institute of Allergy and Infectious Diseasesgrant AI056177 supported this research. The authors thankDr. P. Hauer for supplying monoclonal antibodies againstCPB. We thank Jackie Parker and Jim Cravotta for excellenttechnical assistance.

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