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Ulcerative Enteritis-like Disease Associated with Clostridium sordellii in QuailAuthor(s): Rocio Crespo , Monique Franca , and H. L. ShivaprasadSource: Avian Diseases, 57(3):698-702. 2013.Published By: American Association of Avian PathologistsDOI: http://dx.doi.org/10.1637/10485-010813-Case.1URL: http://www.bioone.org/doi/full/10.1637/10485-010813-Case.1

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Case Report—

Ulcerative Enteritis-like Disease Associated with Clostridium sordellii in Quail

Rocio Crespo,AD Monique Franca,B and H. L. ShivaprasadC

AAvian Health and Food Safety Laboratory, Washington Animal Disease Diagnostic Laboratory, Washington State University, 2607 West Pioneer,Puyallup, WA 98371

BPoultry Diagnostic and Research Center, College of Veterinary Medicine, University of Georgia, 501 D.W. Brooks Drive, Athens, GA 30602CCalifornia Animal Health and Food Safety Laboratory System, Tulare Branch, University of California–Davis, 18830 Road 112,

Tulare, CA 93274

Received 17 January 2013; Accepted 27 March 2013; Published ahead of print 9 April 2013

SUMMARY. A natural outbreak of ulcerative enteritis-like disease associated with Clostridium sordellii was diagnosed in twocommercial quail flocks. Clinical signs in the quail included anorexia, weakness, and increased mortality in the flocks. Lesions in theintestine were characterized by ulcers covered with fibrinonecrotic exudate in the small intestine and occasional hemorrhages. Therewere also multifocal pale areas of necrosis in the liver. Clostridium sordellii was isolated from the intestine and liver. A retrospectivestudy of avian cases submitted to the California Animal Health and Food Safety Laboratories revealed that C. sordellii had beenisolated in 45 avian submissions, most commonly in chickens and turkeys. In most of these cases the birds were diagnosed withnecrotic enteritis, with or without hepatitis. Clostridium sordellii has occasionally been associated with gangrenous dermatitis inpoultry, but this is the first report of enteritis in an avian species.

RESUMEN. Reporte de Caso—Enfermedad similar a la enteritis ulcerativa asociada con Clostridium sordellii en codornices.Se diagnostico un brote natural de una enfermedad similar a enteritis ulcerativa asociada con Clostridium sordellii en dos parvadas

comerciales de codornices. Los signos clınicos en las codornices incluyeron anorexia, debilidad y mortalidad aumentada en lasparvadas. Las lesiones en el intestino estuvieron caracterizadas por ulceras cubiertas con exudado fibrinonecrotico en el intestinodelgado y hemorragias ocasionales. En el hıgado se observaron areas palidas de necrosis. Se aislo Clostridium sordellii del intestino ydel hıgado. Un estudio retrospectivo de los casos aviares remitidos a los Laboratorios de Salud Animal e Inocuidad de los Alimentosde California, revelo que se habıa aislado C. sordellii en 45 casos aviares, mas comunmente en pollos y pavos. En la mayorıa de loscasos, las aves fueron diagnosticadas con enteritis necrotica, con o sin la presencia de hepatitis. Ocasionalmente, Clostridium sordelliiha sido asociado con dermatitis gangrenosa en las aves comerciales, pero este es el primer reporte de enteritis en una especie aviar.

Key words: Clostridium sordellii, enteritis, quail

Abbreviations: CAHFS 5 California Animal Health and Food Safety Laboratories; H&E 5 hematoxylin and eosin;PEA 5 phenyl ethyl alcohol

Ulcerative enteritis is an acute bacterial infection of chickens,turkeys, young quail, and other upland game birds. It ischaracterized by sudden onset and rapidly increasing mortality.The disease is caused by Clostridium colinum (17). It is also called‘‘quail disease’’ because it is especially severe in quail, in which it ischaracterized by sudden onset, rapid spread within a flock, andmortality sometimes as high as 100% (5,17). Ulcerative enteritis hasalso been associated with Clostridium perfringens in quail (13).Lesions are characterized by multiple ulcers throughout the intestinaltract, including ceca and foci of necrosis, and inflammation in theliver (5,17). These ulcers can often perforate the wall of the intestinecausing peritonitis (17).

A disease clinically and pathologically indistinguishable fromulcerative enteritis but associated with C. sordellii, without theinvolvement of either C. perfringens or C. colinum, has never beendescribed in birds. In commercial poultry, C. sordellii has beenoccasionally associated with gangrenous dermatitis in turkeys (7) butit has never been reported in cases of enteritis in birds. This casereport describes two natural outbreaks of ulcerative enteritis-likedisease in flocks of quail associated with C. sordellii. The case alsoprovides a brief retrospective study.

CASE REPORT

Flock history. There were two separate outbreaks of ulcerativeenteritis-like disease on different quail farms. One flock consisted ofapproximately 300, 20-wk-old bobwhite quail (Colinus virginianus)raised on a dirt floor in a barn with rice hulls as litter. Fifty of thesequail died over a 3-wk period with a history of anorexia, weakness,diarrhea, and death. The other flock consisted of approximately5000 multiple quail species on the farm including bobwhite,Tennessee red (C. virginianus mute variant), and California Valley(Callipepla californica). Sixteen out of 1000 California Valley quaildied with a similar history as above. Other species of quail (bobwhiteand Tennessee red) were also affected.

Necropsy and pathology. One live and two dead bobwhite quailfrom the first flock and three dead California Valley quail from thesecond flock were submitted to the Fresno branch of the CaliforniaAnimal Health and Food Safety Laboratories (CAHFS) inDecember 2008 and in February 2009, respectively. The live birdwas humanely euthanatized with carbon dioxide. All birds examinedfrom both flocks had multiple to numerous raised, dry, pale tanplaques or fibrinonecrotic exudate that extended as deep ulcers intothe serosa, primarily in their small intestine (Fig. 1). The contents ofthe affected intestine were greenish-brown mixed with flecks offibrin. Two birds from the first and one from the second submissionDCorresponding author. E-mail: crespor@wsu.edu

AVIAN DISEASES 57:698–702, 2013

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had distended intestines and the contents were brownish and watery.In one bird from the second submission, there was also free blood inthe intestinal lumen. No coccidia were identified on direct smears ofthe intestinal mucosa nor were nematode eggs observed. Ulcers werealso occasionally observed in the large intestine. The livers weremildly enlarged and there were many pale white, nonraised, pinpointfoci randomly scattered throughout in two birds (Fig. 2).

Histopathology. Samples of esophagus, crop, proventriculus,gizzard, small and large intestine, liver, pancreas, and spleen from allthe birds received were collected and fixed in 10% neutral bufferedformalin for a minimum of 24 hr. In addition, samples of brain,spinal cord, conjunctiva, trachea, lung, heart, kidney, bursa ofFabricius, thymus, skeletal muscle, bone, bone marrow, thyroid,skin, adrenals, eyes, and ears from a few of these birds were alsocollected and fixed in 10% neutral buffered formalin for a minimumof 24 hr. All tissues were dehydrated in graded alcohols to xylene,embedded in paraffin, sectioned at 4 mm, stained with hematoxylinand eosin (H&E), and examined by light microscopy. In addition,selected sections of intestine and liver were Gram-stained bymodified Brown-Hopps (6).

Histologic examination of the intestine revealed severe, multifocalnecrosis of the mucosa with fibrinosuppurative inflammationassociated with large numbers of rod-shaped gram-positive bacteria(Fig. 3). The ulcers were walled off by large numbers of fibroblastsnear the serosa and muscular layers. Often, these ulcers extendeddeep in to the mucosa, muscularis mucosae, muscular layers, andserosa causing mild to severe peritonitis. No parasites, most notablycoccidia, were observed in the intestine. In addition, there were

random areas of acute, coagulative necrosis of hepatocytes and fibrinexudation mixed with heterophils (Fig. 4). Intralesional, rod-shaped,Gram-positive bacteria were seen occasionally in the liver.

Bacteriology. Four intestine and two liver samples from a total ofsix birds (one from each submission) received were asepticallycollected and cultured for anaerobic bacteria on prereduced phenylethyl alcohol (PEA) sheep blood agar and Brucella base 5% sheepblood agar prereduced, anaerobically sterilized media supplementedwith vitamin K and hemin (Anaerobe Systems, Morgan Hill, CA).Intestinal samples were also cultured in chopped meat carbohydratemedium (Anaerobe Systems), incubated at 37 C for 24 hr, andplated onto PEA agar and Brucella 5% sheep blood agar. Inaddition, samples from four livers and six intestines were culturedonto a specialized medium containing 8% horse plasma specificallydesigned for the isolation of C. colinum (13). These plates wereincubated under anaerobic conditions at 37 C for up to 4 days. Theplates were examined for bacterial growth every 24 hr. SuspectClostridium sp. isolates were further examined by Gram stain, colonymorphology, and aerotolerance on chocolate agar plates (at 37 C in5–10% CO2 for 24 hr).

Additionally, three livers were plated on 5% sheep blood andMacConkey agars (Remel, Lenexa, KS) and incubated at 37 C with5% CO2 for a minimum of 48 hr. Intestines were pooled and theircontents were selectively enriched in selenite enrichment broth at42 C for 18–24 hr. After enrichment, selenite broth was plated on

Fig. 1. Photograph of the small intestine with numerous raised, dry,pale tan plaques or fibrino-necrotic exudate that extended as deep ulcersinto the serosa and are visible through the intestinal wall (arrows). Theliver, with a few pale foci, and the proventriculus (top left) have beendisplaced to the side to allow visualization of small intestine.

Fig. 2. Photograph of a liver showing numerous pale white,nonraised, pinpoint foci randomly scattered throughout.

C. sordellii enteritis in quail 699

brilliant green and XLT4 agars (Remel) and incubated at 37 C for anadditional 24 hr.

After 48 hr of anaerobic incubation, the liver and intestinalcultures showed large, lobulated, irregular, swarming and flatcolonies with serpentine edges. On Gram-staining of the culturesfrom both the liver and intestine, the organisms appeared as large,gram-positive rods with rounded edges and central spores (Fig. 5).The bacteria were identified as C. sordellii by Rap ID Ana II System(Remel). The bacteria were indole- and lecithinase positive, rapidurease positive, and catalase negative; the bacteria were also sensitiveto kanamycin and vancomycin antibiotics. In addition, C.perfringens was isolated from one of the affected intestines. Noaerobic bacteria from the livers were isolated nor were salmonellaefrom the intestine.

Retrospective study. A retrospective search of the CAHFSdatabase found 46 cases, between 1990 and 2012, diagnosed fromvarious avian species in which C. sordellii had been isolated. Fifteencases were from chickens, 10 from turkeys, 10 from ostriches, sixfrom psittacines, three from quail, and one each from a swan and agoose. The most common complaint in all the cases was that thebirds appeared sick for 2 days before death. The most commondiagnoses in chickens and turkeys were necrotic-ulcerative enteritisand in other birds necrotic enteritis and occasionally hepatitis.

Clostridium sordellii were also isolated from five cases of navelinfections and from two cases of gangrenous dermatitis in chickens.

DISCUSSION

Severe lesions in the intestine and liver associated with gram-positive bacilli accounted for the clinical signs and death of thesequail. The clinical signs and lesions in these birds resembledulcerative enteritis. Ulcerative enteritis, also called quail disease, isone of the most common and important diseases of quail caused bythe bacterium C. colinum (17); however, extensive anaerobicculturing of the intestine and liver from the quail yielded purecultures of C. sordellii. Only one intestinal sample had a mixedculture of C. sordellii and C. perfringens. Clostridium colinum was notisolated from the liver or the intestine.

Clostridium sordellii is a gram-positive, anaerobic, and spore-forming bacillus that produces exotoxins (9). This organism isubiquitous and occurs in soil, water, sewage, and the intestinal tractsof animals and humans (10). Clostridium sordellii has beenrecognized as a pathogenic agent in humans, cattle, and sheep. Inthese species it has been primarily associated with wound infections(8). Colitis and, more recently, septic endometritis and septic shock

Fig. 3. Microphotograph of intestine showing severe, multifocal necrosis of the mucosa with fibrino-suppurative inflammation associated withlarge numbers of rod-shaped bacteria. H&E. Bar 5 20 mm. Insert: Detail of intestinal necrotic debris with numerous Gram-positive bacteria. Gramstain. Bar 5 10 mm.

700 R. Crespo et al.

associated with C. sordellii have been reported in humans (1,3,4,12).A few reports suggest that C. sordellii is responsible forenterotoxemia-like syndromes in cattle and sheep (8,15). Incommercial poultry, C. sordellii has been occasionally associatedwith gangrenous dermatitis in turkeys (7) but it has never beenreported as a causative agent of necrotic or ulcerative enteritis.

It is generally known that C. colinum causes lesions throughoutthe intestinal tract, including ceca (17), whereas lesions with necroticenteritis due to C. perfringens are primarily confined to the smallintestine (13,16). In the current case the distribution of the lesions inthe intestinal tract was similar to infections due to C. perfringens.The pathogenesis of the condition described in this article isunknown. Some of the toxins produced by C. sordellii are moreclosely related to Clostridium difficile and differ greatly from alpha-toxin produced by C. perfringens (1,15). On the other hand, recentstudies have demonstrated that alpha-toxin may not be the onlymajor pathologic virulent factor in C. perfringens, and novel toxinssuch as TpeL and NetB may have an important role in inducingnecrotic enteritis (14). TpeL has 39% homology to C. sordellii lethaltoxin (TcsL) (2). Thus, it would be then possible to hypothesize thatthe TcsL in C. sordellii could cause similar lesions to the TpeL toxinin C. perfringens. Furthermore, NetB is mostly found in C.perfringens associated with necrotic enteritis outbreaks but it is

uncommon in C. perfringens isolates from normal chickens (11). It isunknown if C. sordellii is capable of producing NetB or a NetB-liketoxin. It is also unknown whether C. sordellii is a normal intestinalflora of birds and, in particular, of quail. However, C. sordellii wasfound in 46 avian cases submitted to CAHFS in the last 20 yr. Mostof these cases were diagnosed with enteritis, with or withouthepatitis, followed by navel infection and gangrenous dermatitis.These findings suggest that C. sordellii may be normal intestinal floraand, like in other animals, they remain dormant for the life of theanimal unless presented with conditions appropriate for germinationand growth. More studies are needed to determine the pathogenesisand toxins produced by C. sordellii in birds. Diagnosticians andpractitioners should consider C. sordellii in their differentials whenulcerative or necrotic enteritis is encountered in quail.

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Fig. 4. Microphotograph of the liver showing random, multifocalareas of acute coagulative necrosis of hepatocytes and fibrin exudationmixed with heterophils. Note large number of rod-shaped bacteria(arrows) in the necrotic areas of the liver. H&E. Bar 5 20 mm.

Fig. 5. Photograph showing typical morphology of Clostridiumsordellii. The bacteria are gram-positive rods with rounded edges andcentral spores. Gram stain. Bar 5 10 mm.

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