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Avian Pathology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cavp20
Clostridial co-infection episodes in commercial laying hens a
a
a
a
b
a
G. Berto , F. Agnoletti , I. Drigo , E. Tonon , M. Vascellari , V. Fracas & L. Bano
a
a
Veterinary Diagnostic Laboratory, Istituto Zooprofilattico Sperimentale delle Venezie, Treviso, Italy b
Laboratory of Histopathology, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy Accepted author version posted online: 13 Mar 2015.
Click for updates To cite this article: G. Berto, F. Agnoletti, I. Drigo, E. Tonon, M. Vascellari, V. Fracas & L. Bano (2015): Clostridial coinfection episodes in commercial laying hens, Avian Pathology, DOI: 10.1080/03079457.2015.1028333 To link to this article: http://dx.doi.org/10.1080/03079457.2015.1028333
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Publisher: Taylor & Francis & Houghton Trust Ltd Journal: Avian Pathology DOI: http://dx.doi.org/10.1080/03079457.2015.1028333
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an us
Clostridial co-infection episodes in commercial laying hens
Berto Giacomo¹* ([email protected]) , Agnoletti Fabrizio¹
([email protected]), Drigo Ilenia¹ ([email protected]), Tonon Elena¹
M
([email protected]), Vascellari Marta² ([email protected]), Fracas
ed
Valentina¹ ([email protected]), Bano Luca¹ ([email protected]).
¹ Istituto Zooprofilattico Sperimentale delle Venezie, Veterinary Diagnostic Laboratory,
pt
Vicolo Mazzini 5/4, 31020, Villorba, Treviso, Italy ² Istituto Zooprofilattico Sperimentale
ce
delle Venezie, Laboratory of Histopathology, Viale dell’Università 10, 35020, Legnaro, Padova, Italy
Ac
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Cavp-2015-0029.R1
Short Title: Clostridial co-infection in hens
*Corresponding author: Tel: +39 0422 302302. Fax: +39 0422 421154. E-mail: [email protected]
Received: 6 February 2015
Abstract
The present report describes two outbreaks of serious enteritis in commercial laying hens where Clostridium perfringens (C. perfringens) and Clostridium colinum (C. colinum) were
cr ip t
simultaneously detected. At the age of 44 and 31 weeks, two laying hen flocks showed an
examination revealed intestinal necrotic-hemorrhagic ulcerations and hepatic focal necrosis.
an us
The bacteriological examination yielded the isolation of C. colinum and C. perfringens toxin type A, NetB positive. In one outbreak C. colinum was detected also by PCR in all the intestines of affected birds.
In laying hens C. colinum has never been isolated but only suspected as the causative
M
agent of a slight enteric disease called duodenal focal necrosis. The present case report was
ed
characterized by severe enteritis presumably due to the synergistic effect of C. colinum and C.
ce
Introduction
pt
perfringens.
Bacterial enteritis in laying hens (Gallus gallus) are primarily caused by anaerobic
Ac
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increase of the mortality rate and a worsening of productive performance. Post-mortem
microorganisms belonging to the genera Clostridium and Brachyspira (Hampson et al., 2008).
The most frequent among these pathologies is necrotic enteritis (NE), caused by Clostridium pefringens (C. perfringens), a Gram positive sporogenic anaerobic bacillus that acts as a pathogen through the production of diverse types of toxins. C. perfringens is classified in 5 toxin types (A, B, C, D, E) in relation to the different combinations of major toxins produced. Historically NE in poultry is associated with toxin types A and C, however research
conducted in several countries has demonstrated the implication of only toxin type A characterized by the production of α-toxin (Songer, 1996; Olkowski et al., 2008, Drigo et al, 2009). In the past, α-toxin produced by all the strains of C. perfringens, was thought to be an essential virulence factor for the occurrence of the disease (Al-Shiekhly & Treuescott, 1977), but today it is acknowledged that its presence is not necessary for the development of the
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typical lesions (Keyburn et al., 2006). The toxin considered the most significant in the
The gross lesions and the severity of the clinical signs can be more serious if the strain
an us
involved produces also a toxin belonging to the group “large clostridial cytotoxins”
denominated TpeL (Courdoson et al., 2012). NE generally strikes broilers from 2 to 6 weeks of age, even though reports of full-blown disease exist in commercial pullets and laying hens from 3 to 9 months of age (Kwatra et al., 1976; Barbes et al., 2008; Porter et al., 1998).
M
Another important clostridial disease in poultry is ulcerative enteritis (UE), caused by
ed
Clostridium colinum (C. colinum), which primarily affects the Virginia Quail (Colinus virginianus), in which the pathology often assumes an epidemic nature. The C. colinum
pt
infection has been also reported in chickens even if the disease has not been reproduced in subjects orally inoculated (Berkhoff & Campbell, 1973; Berkoff et al., 1973). Although C.
ce
colinum is considered a primary pathogen for poultry, the combination with other predisposing factors (eg. coccidiosis, Gumboro disease, infective anemia and stress) may
Ac
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etiopathogenesis of NE is a “pore-forming protein” denominated NetB (Keyburn et al., 2008).
worsen the clinical picture (Davis, 1973). In laying hens, C. colinum has been suspected as the causative agent of the so-called duodenal focal necrosis (DFN) although the presence of the pathogen in this disease has never been clearly demonstrated and lesion reproduced
(Baltzely et al., 2008).
Materials and Methods
Case history. Over the course of 2013 in a Hyline commercial laying hen farm, 2 outbreaks of disease characterized by sudden increases of mortality were observed. The first episode occurred in June and involved a group of 44,000 subjects of 47 weeks of age, while the
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second occurred in December in a group of 18,300 subjects of 31 weeks of age.
farmer stated that there was a sudden decrease in productive performance with reduction of
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the egg weight (from 65 gram to 60 gram), decreased food consumption (from 106 to 93
gram/ head/ day) and an average mortality rate of 0.5%. In the 31-week old group, there were also animals with gastrointestinal symptoms localized in only one section of the shed. The principal symptom was characterized by diarrhea with the presence of not completely
M
digested food.
ed
Thirteen animal carcasses (4 from group 1 and 9 from group 2) were sent to the
pt
veterinary diagnostic laboratory of Treviso for diagnostic purposes
Bacteriological isolation and identification. Liver of one bird and intestinal content of two
ce
birds of the first outbreak with macroscopic lesions were sent for bacteriological examination. In addition, bacteriological examination was conducted from the intestinal ulcers of four
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Both groups of animals were cage-raised in different sheds of the same farm. The
subjects of the second outbreak. The samples aseptically collected from liver and intestine
were inoculated in Columbia agar (CA) with 5% of sheep blood, (Becton Dickinson, USA), perfringens agar base (Oxoid, Basingstone, UK) with 5% of sheep blood (PAB), cooked meat medium (CMM) (Oxoid, Basingstone, UK) and eosin methylene blue (EMB) (Oxoid, Basingstone, UK). The CA and EMB plates were incubated for 24 hours at 37± 1 ° C in
aerobiosis, while PAB, CMM and a second CA plate were incubated at the same temperature
in anaerobiosis for 24-48 hours. The culture media were inspected after 24 to 48 hours. The identification of isolates was carried out through MALDI-TOF MS (Maldi Biotyper, Bruker Daltonics).
Histopathology and parasitology. Samples of liver with macroscopically evident lesions
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were subjected to virological and histopathological exams. The virological exam was
eggs and conventional cell lines (chick embryo hepatocytes). For the histological exam,
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portions of liver and intestine were fixed in 10% buffered formalin, included in paraffin and sectioned at 4 µm. The sections were then stained with hematoxylin and eosin stain and the preparations were observed under an optical microscope.
A parasitological exam was also conducted through direct microscopic observation
M
(X100) of intestinal scrapings made on the small intestine, cecum and at the level of the
ed
macroscopic lesions.
pt
PCR detection of C. colinum and toxin typing of C. perfringens. One colony of C. perfringens isolated from each tested sample (picked up from PAB or CA) was toxinotyped
ce
through multiplex-PCR and the NetB toxin encoding gene was also investigated (Yoo et al., 1997, Keyburn et al., 2008). In birds sent in December, from the intestinal contents, the
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conducted through electronic microscopy, isolation in embryonated specific pathogen free
presence of C. colinum was also investigated by PCR in the intestinal tracts with evident lesions consistent with ulcerative enteritis (Bano et al.,2008). Results In all sent carcasses post-mortem examination revealed round-shaped intestinal lesions of variable diameter (0.5-3 mm), with necrotic-hemorrhagic center, raised margins and occasionally coalescent. The ulcerations with the largest diameters were also visible through
the serous membrane of the intestine (Figure 1), while there were round necrotic areas in some tracts of the mucous membrane measuring 1-2 cm in diameter (Figure 2a, Figure 2b). The cecum lumen were enlarged by caseous material of hemorrhagic aspect composed by fibrin and necrotic debris, originating from ulcerations disseminated on the mucous membrane and visible after washing (Figure 3). A focal necrotic hepatitis was observed in
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two subjects of the first flock, while the spleen was generally non-reactive, pallid but with
At a histological level, necrotic enteritis was detected with various stages of erosion of
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the mucous surface, associated with the presence of bacterial aggregates and diffuse
lymphoplasmacytic infiltration (Figure 4). The liver was affected by multifocal heterophilicnecrotic hepatitis associated with bacterial aggregates and diffuse steatosis of the hepatocytes (Figure 5). Results concerning PCR and bacteriological examination are summarized in table
M
1. Bacteriological examination revealed high levels of toxin type A C. perfringens isolated
ed
from the intestine of subjects belonging to both groups. Two strains of C. perfringens isolated over the course of the first outbreak were also positive for the NetB toxin encoding gene.
pt
None of C. perfringens isolated from second group were positive for the NetB encoding gene. From the liver of one subject with necrotic lesions it was possible to isolate C. colinum in CA
ce
incubated in anaerobic conditions. C. colinum was identified at species level through MALDITOF MS (score 2.106) as well as through PCR performed directly from the isolate, while no
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hemorrhagic suffusions on the surface.
direct PCR were conducted on the intestinal contents of subjects belonging to the first submission. In the intestines of all examined birds of the second outbreak, both C. perfringens and C. colinum were detected. In the animals belonging to second group C. colinum were not isolated but only detected by PCR from the intestinal contents. The virological exams were negative, while the parasitological exam detected modest presence of coccidia oocysts only in three out of the 12 subjects examined.
Discussion
Necrotic enteritis is a typical disease of meat chickens that is becoming more and more common in laying hens. The different susceptibility to NE in the aforementioned productive
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categories (broiler and laying hen) can be sought in the possibility that predisposing factors
sensitivity related to the age of the host. However, the lesions that usually are observed in NE
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outbreaks in laying hens do not reach the same serious level as described in the present report. In both the affected groups investigated in this study, the simultaneous presence of C. perfringens and C. colinum was detected. Some isolated strains in the first group harbored the NetB encoding gene, while the presence of C. colinum was not initially suspected and
M
therefore not investigated directly from the intestinal lesions. The unexpected isolation of C.
ed
colinum from the liver permitted a retrospective diagnosis of a possible case of UE. In the second case, which occurred 6 months later in another group of laying hens, the presence of
pt
C. colinum was investigated directly through PCR, demonstrating the presence of C. colinum in all affected subjects.
ce
In the presented clinical case it is impossible to establish with certainty which of the
two Clostridium species has the predominant etiological role, but the isolation of C. colinum
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(e.g. coccidiosis) can contribute to the development of the pathology, rather than in a different
in the liver presenting the classic necrotic lesions is nevertheless significant, as these are usually detected in UE in other animal species. The only reports of UE in laying hens refer to a focal necrotic duodenitis in which C. colinum has never been isolated but only identified
through molecular methods. A simultaneous C. colinum and C. perfringens co-infection in laying hens has never been reported. However, Beltran-Alcrudo et al. (2008) reported
isolation of both bacteria in a case of ulcerative enteritis in quails, considering C. colinum as primary agent of the pathology and attributing C. perfringens a secondary role. Following the diagnosis of intestinal clostridiosis the animals were treated for 5 days with tylosin in drinking water (50 g/100 L). It is well known that the strains of C. perfringens isolated from chicken are usually sensitive to tylosin but the lack of selective growth media
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for C. colinum makes the isolation from particularly contaminated organs difficult, as well as
Gholamiandehkordi et al., 2009). The fact that after treatment there was a remission in
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pt
ed
M
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symptoms leads to the conclusion that C. colinum can also be susceptible to this molecule.
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the execution of tests to evaluate the susceptibility to antimicrobials (Martel et al., 2004;
References
Al-Shiekhly F, Truescott RB. (1977). The pathology of necrotic enteritis of chickens following infusion of broth cultures of Clostridium perfringens into the duodenum.
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Avian Diseases, 21, 230-240
following infusion of crude toxins of Clostridium perfringens into the duodenum.
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Avian Diseases, 21, 241-255
Baltzely TA, Dunham SM, Lago F, Rehberger TG, Siragusa GR. (2008). Molecular pathogenesis markers of focal duodenal necrosis in layer hens. Proceedings of the 80th Northeastern conference on Avian diseases.
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Bano L, Drigo I, Macklin KS, Martin RS, Miller RS, Norton RA, Oyarzabal OA, Bilgili SF.
ed
(2008). Development of a polymerase chain reaction assay for specific identification of Clostridium colinum. Avian Pathology, 37, 179-181
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Barnes HJ, Wages DP, Opengart K. (2008). Clostridial disease. In YM Saif, AM Fadly, JR Glisson, LR McDougald, LK Nolan (Eds.). Diseases of Poultry, 12th Edn (pp 865-
ce
879). Blackwell Publishing, London.
Beltran-Alcrudo D, Cardona C, McLellan L, Reimers N, Charlton B. (2008). A persistent
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Al-Shiekhly F, Truescott RB. (1977). The pathology of necrotic enteritis of chickens
outbreak of ulcerative enteritis in bobwhite quail (Colinus virginianus). Avian
Diseases, 52, 531-536
Berkhoff GA, Campbell SG. (1973). Etiology and pathogenesis of ulcerative enteritis (quail disease). The experimental disease. Avian Diseases, 18, 205-2012
Berkhoff GA, Campbell SG, Naylor HB. (1973). Etiology and pathogenesis of ulcerative enteritis (quail disease). Isolation of the causative anaerobe. Avian Diseases, 18, 186194 Coursodon CF, Glock RD, Moore KL, Cooper KK, Songer JG. (2012). TpeL-producing strains of Clostridium perfringens type A are higly virulent for broiler chicks.
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Anaerobe, 18, 117-121
factors. Poultry Scence, 52, 1283-1287
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Drigo I, Agnoletti F, Bacchin C, Guolo A, Cocchi M, Bonci M, Bano L. ( 2009). Diffusion of Clostridium perfringens NetB positive strain in healthy and diseased chicken. Italian Journal of Animal Science, 8, 761-764
Gholamiandehkordi A, Eeckaut V., Lanckriet A, Timbermont L, Bjerrum L, Ducatelle R,
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Haesebrouck F, Van Immerseel F. (2009). Antimicrobial resistance in Clostridium
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perfringens isolates from broilers in Belgium. Veterinary Research Communication, 33, 1031-1037
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Hampson DJ, Swayne DE. (2008). Avian intestinal Spirochetosis. In YM Saif, AM Fadly, JR Glisson, LR McDougald, LK Nolan (Eds.). Diseases of Poultry, 12th Edn (pp. 922-
ce
940). Blackwell Publishing, London.
Keyburn AL, Sheedy SA, Ford ME, Williamson M M, Award M M, Rood JI, Moore RJ
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Davis RB. (1973). Ulcerative enteritis in chickens: coccidiosis and stress as predisposing
(2006). Alpha-toxin of Clostridium perfringens is not an essential virulence factor in necrotic enteritis in chickens. Infecion and Immunity, 74, 6496-6500
Keyburn AL, Boyce JD, Vaz P, Bannma TL, Ford ME, Parker D, Di Rubbo A, Rood JI, Moore RJ. (2008). NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens. PLoS Pathogen, 4, 0001-0011
Kwatra MS, Chaudhury B. (1976). A presumptive diagnosis in the fowl (Gallus gallus domesticus) from Assam (India). Avian Diseases, 20, 401-406 Martel A, Devriese LA, Cauwerts K, De Gussem K., Decostere A., Haesebrouck F. (2004) Susceptibility of Clostidium perfringens stains from broilers chickens to antibiotics and anticoccidials. Avian Pathology, 33, 3-7
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Olkowski AA, Wojnaroicz C, Chirino-Trejo M, Laarveld B, Sawicki G. (2008). Sub-clinical
structural and molecular changes in the intestinal tissue. Research in Veterinary
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Science, 85, 543-553
Porter RE Jr. (1998). Bacterial enteritides of poultry. Poultry Science, 77, 1159-1165 Songer JG. (1996). Clostridial enteric disease of domestic animals. Clinical Microbiology Reviews, 9, 216-234
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Yoo HS, Lee SU, Park YH. (1997). Molecular typing and epidemiological survey of
ed
prevalence of Clostridium perfringens types by multiplex PCR. Journal of Clinical
ce
pt
Microbiology, 35, 228-232
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necrotic enteritis in broiler chickens: novel etiological consideration based on ultra-
Figure Legends
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Figure 1. Intestinal ulceration visible through the serous membrane of the intestine.
Figure 2a. Intestinal ulceration visible from mucosal surface. Note the extension of the
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pathologic process
pt
ce
Ac ed cr ip t
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Figure 2b. Detail. Intestinal ulceration
pt
ce
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Figure 3. Ceca. Advanced ulcers filled with diphtheritic, necrotic debris.
Figure 4. Intestine. Uniform diffuse coagulation necrosis of the mucosa, diffuse
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lymphoplasmacytic infiltration and bacterial aggregates x20
Figure 5. Liver. Multifocal heterophilic-necrotic hepatitis associated with bacterial
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aggregates and diffuse steatosis of the hepatocytes X40
Table 1. Results of bacteriological and biomolecular examinations Outbreak
Subject
1 1a
Organ
Isolate
C.perfringens C. pefringens
PCR
Toxinotype
NetB gene
C. colinum
liver
C. colinum
-
-
positive
intestine
C.
A
positive
ntc
A
positive
ntc
2
intestine
C. perfringens
intestine
C.
2b
A
perfringens 4
intestine
C.
A
8
intestine
negative
positive
negative
positive
an us
perfringens C.
A
negative
positive
A
negative
positive
perfringens 9
intestine
C.
perfringens b
c
M
June 2013; Dicember 2013; not texted
ce
pt
ed
a
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3
cr ip t
perfringens
Avian Pathology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cavp20
Clostridial co-infection episodes in commercial laying hens a
a
a
a
b
a
G. Berto , F. Agnoletti , I. Drigo , E. Tonon , M. Vascellari , V. Fracas & L. Bano
a
a
Veterinary Diagnostic Laboratory, Istituto Zooprofilattico Sperimentale delle Venezie, Treviso, Italy b
Laboratory of Histopathology, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy Accepted author version posted online: 13 Mar 2015.
Click for updates To cite this article: G. Berto, F. Agnoletti, I. Drigo, E. Tonon, M. Vascellari, V. Fracas & L. Bano (2015): Clostridial coinfection episodes in commercial laying hens, Avian Pathology, DOI: 10.1080/03079457.2015.1028333 To link to this article: http://dx.doi.org/10.1080/03079457.2015.1028333
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Publisher: Taylor & Francis & Houghton Trust Ltd Journal: Avian Pathology DOI: http://dx.doi.org/10.1080/03079457.2015.1028333
cr ip t
an us
Clostridial co-infection episodes in commercial laying hens
Berto Giacomo¹* ([email protected]) , Agnoletti Fabrizio¹
([email protected]), Drigo Ilenia¹ ([email protected]), Tonon Elena¹
M
([email protected]), Vascellari Marta² ([email protected]), Fracas
ed
Valentina¹ ([email protected]), Bano Luca¹ ([email protected]).
¹ Istituto Zooprofilattico Sperimentale delle Venezie, Veterinary Diagnostic Laboratory,
pt
Vicolo Mazzini 5/4, 31020, Villorba, Treviso, Italy ² Istituto Zooprofilattico Sperimentale
ce
delle Venezie, Laboratory of Histopathology, Viale dell’Università 10, 35020, Legnaro, Padova, Italy
Ac
Downloaded by [University of Nebraska, Lincoln] at 04:56 03 April 2015
Cavp-2015-0029.R1
Short Title: Clostridial co-infection in hens
*Corresponding author: Tel: +39 0422 302302. Fax: +39 0422 421154. E-mail: [email protected]
Received: 6 February 2015
Abstract
The present report describes two outbreaks of serious enteritis in commercial laying hens where Clostridium perfringens (C. perfringens) and Clostridium colinum (C. colinum) were
cr ip t
simultaneously detected. At the age of 44 and 31 weeks, two laying hen flocks showed an
examination revealed intestinal necrotic-hemorrhagic ulcerations and hepatic focal necrosis.
an us
The bacteriological examination yielded the isolation of C. colinum and C. perfringens toxin type A, NetB positive. In one outbreak C. colinum was detected also by PCR in all the intestines of affected birds.
In laying hens C. colinum has never been isolated but only suspected as the causative
M
agent of a slight enteric disease called duodenal focal necrosis. The present case report was
ed
characterized by severe enteritis presumably due to the synergistic effect of C. colinum and C.
ce
Introduction
pt
perfringens.
Bacterial enteritis in laying hens (Gallus gallus) are primarily caused by anaerobic
Ac
Downloaded by [University of Nebraska, Lincoln] at 04:56 03 April 2015
increase of the mortality rate and a worsening of productive performance. Post-mortem
microorganisms belonging to the genera Clostridium and Brachyspira (Hampson et al., 2008).
The most frequent among these pathologies is necrotic enteritis (NE), caused by Clostridium pefringens (C. perfringens), a Gram positive sporogenic anaerobic bacillus that acts as a pathogen through the production of diverse types of toxins. C. perfringens is classified in 5 toxin types (A, B, C, D, E) in relation to the different combinations of major toxins produced. Historically NE in poultry is associated with toxin types A and C, however research
conducted in several countries has demonstrated the implication of only toxin type A characterized by the production of α-toxin (Songer, 1996; Olkowski et al., 2008, Drigo et al, 2009). In the past, α-toxin produced by all the strains of C. perfringens, was thought to be an essential virulence factor for the occurrence of the disease (Al-Shiekhly & Treuescott, 1977), but today it is acknowledged that its presence is not necessary for the development of the
cr ip t
typical lesions (Keyburn et al., 2006). The toxin considered the most significant in the
The gross lesions and the severity of the clinical signs can be more serious if the strain
an us
involved produces also a toxin belonging to the group “large clostridial cytotoxins”
denominated TpeL (Courdoson et al., 2012). NE generally strikes broilers from 2 to 6 weeks of age, even though reports of full-blown disease exist in commercial pullets and laying hens from 3 to 9 months of age (Kwatra et al., 1976; Barbes et al., 2008; Porter et al., 1998).
M
Another important clostridial disease in poultry is ulcerative enteritis (UE), caused by
ed
Clostridium colinum (C. colinum), which primarily affects the Virginia Quail (Colinus virginianus), in which the pathology often assumes an epidemic nature. The C. colinum
pt
infection has been also reported in chickens even if the disease has not been reproduced in subjects orally inoculated (Berkhoff & Campbell, 1973; Berkoff et al., 1973). Although C.
ce
colinum is considered a primary pathogen for poultry, the combination with other predisposing factors (eg. coccidiosis, Gumboro disease, infective anemia and stress) may
Ac
Downloaded by [University of Nebraska, Lincoln] at 04:56 03 April 2015
etiopathogenesis of NE is a “pore-forming protein” denominated NetB (Keyburn et al., 2008).
worsen the clinical picture (Davis, 1973). In laying hens, C. colinum has been suspected as the causative agent of the so-called duodenal focal necrosis (DFN) although the presence of the pathogen in this disease has never been clearly demonstrated and lesion reproduced
(Baltzely et al., 2008).
Materials and Methods
Case history. Over the course of 2013 in a Hyline commercial laying hen farm, 2 outbreaks of disease characterized by sudden increases of mortality were observed. The first episode occurred in June and involved a group of 44,000 subjects of 47 weeks of age, while the
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second occurred in December in a group of 18,300 subjects of 31 weeks of age.
farmer stated that there was a sudden decrease in productive performance with reduction of
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the egg weight (from 65 gram to 60 gram), decreased food consumption (from 106 to 93
gram/ head/ day) and an average mortality rate of 0.5%. In the 31-week old group, there were also animals with gastrointestinal symptoms localized in only one section of the shed. The principal symptom was characterized by diarrhea with the presence of not completely
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digested food.
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Thirteen animal carcasses (4 from group 1 and 9 from group 2) were sent to the
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veterinary diagnostic laboratory of Treviso for diagnostic purposes
Bacteriological isolation and identification. Liver of one bird and intestinal content of two
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birds of the first outbreak with macroscopic lesions were sent for bacteriological examination. In addition, bacteriological examination was conducted from the intestinal ulcers of four
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Both groups of animals were cage-raised in different sheds of the same farm. The
subjects of the second outbreak. The samples aseptically collected from liver and intestine
were inoculated in Columbia agar (CA) with 5% of sheep blood, (Becton Dickinson, USA), perfringens agar base (Oxoid, Basingstone, UK) with 5% of sheep blood (PAB), cooked meat medium (CMM) (Oxoid, Basingstone, UK) and eosin methylene blue (EMB) (Oxoid, Basingstone, UK). The CA and EMB plates were incubated for 24 hours at 37± 1 ° C in
aerobiosis, while PAB, CMM and a second CA plate were incubated at the same temperature
in anaerobiosis for 24-48 hours. The culture media were inspected after 24 to 48 hours. The identification of isolates was carried out through MALDI-TOF MS (Maldi Biotyper, Bruker Daltonics).
Histopathology and parasitology. Samples of liver with macroscopically evident lesions
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were subjected to virological and histopathological exams. The virological exam was
eggs and conventional cell lines (chick embryo hepatocytes). For the histological exam,
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portions of liver and intestine were fixed in 10% buffered formalin, included in paraffin and sectioned at 4 µm. The sections were then stained with hematoxylin and eosin stain and the preparations were observed under an optical microscope.
A parasitological exam was also conducted through direct microscopic observation
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(X100) of intestinal scrapings made on the small intestine, cecum and at the level of the
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macroscopic lesions.
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PCR detection of C. colinum and toxin typing of C. perfringens. One colony of C. perfringens isolated from each tested sample (picked up from PAB or CA) was toxinotyped
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through multiplex-PCR and the NetB toxin encoding gene was also investigated (Yoo et al., 1997, Keyburn et al., 2008). In birds sent in December, from the intestinal contents, the
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conducted through electronic microscopy, isolation in embryonated specific pathogen free
presence of C. colinum was also investigated by PCR in the intestinal tracts with evident lesions consistent with ulcerative enteritis (Bano et al.,2008). Results In all sent carcasses post-mortem examination revealed round-shaped intestinal lesions of variable diameter (0.5-3 mm), with necrotic-hemorrhagic center, raised margins and occasionally coalescent. The ulcerations with the largest diameters were also visible through
the serous membrane of the intestine (Figure 1), while there were round necrotic areas in some tracts of the mucous membrane measuring 1-2 cm in diameter (Figure 2a, Figure 2b). The cecum lumen were enlarged by caseous material of hemorrhagic aspect composed by fibrin and necrotic debris, originating from ulcerations disseminated on the mucous membrane and visible after washing (Figure 3). A focal necrotic hepatitis was observed in
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two subjects of the first flock, while the spleen was generally non-reactive, pallid but with
At a histological level, necrotic enteritis was detected with various stages of erosion of
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the mucous surface, associated with the presence of bacterial aggregates and diffuse
lymphoplasmacytic infiltration (Figure 4). The liver was affected by multifocal heterophilicnecrotic hepatitis associated with bacterial aggregates and diffuse steatosis of the hepatocytes (Figure 5). Results concerning PCR and bacteriological examination are summarized in table
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1. Bacteriological examination revealed high levels of toxin type A C. perfringens isolated
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from the intestine of subjects belonging to both groups. Two strains of C. perfringens isolated over the course of the first outbreak were also positive for the NetB toxin encoding gene.
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None of C. perfringens isolated from second group were positive for the NetB encoding gene. From the liver of one subject with necrotic lesions it was possible to isolate C. colinum in CA
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incubated in anaerobic conditions. C. colinum was identified at species level through MALDITOF MS (score 2.106) as well as through PCR performed directly from the isolate, while no
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hemorrhagic suffusions on the surface.
direct PCR were conducted on the intestinal contents of subjects belonging to the first submission. In the intestines of all examined birds of the second outbreak, both C. perfringens and C. colinum were detected. In the animals belonging to second group C. colinum were not isolated but only detected by PCR from the intestinal contents. The virological exams were negative, while the parasitological exam detected modest presence of coccidia oocysts only in three out of the 12 subjects examined.
Discussion
Necrotic enteritis is a typical disease of meat chickens that is becoming more and more common in laying hens. The different susceptibility to NE in the aforementioned productive
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categories (broiler and laying hen) can be sought in the possibility that predisposing factors
sensitivity related to the age of the host. However, the lesions that usually are observed in NE
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outbreaks in laying hens do not reach the same serious level as described in the present report. In both the affected groups investigated in this study, the simultaneous presence of C. perfringens and C. colinum was detected. Some isolated strains in the first group harbored the NetB encoding gene, while the presence of C. colinum was not initially suspected and
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therefore not investigated directly from the intestinal lesions. The unexpected isolation of C.
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colinum from the liver permitted a retrospective diagnosis of a possible case of UE. In the second case, which occurred 6 months later in another group of laying hens, the presence of
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C. colinum was investigated directly through PCR, demonstrating the presence of C. colinum in all affected subjects.
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In the presented clinical case it is impossible to establish with certainty which of the
two Clostridium species has the predominant etiological role, but the isolation of C. colinum
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(e.g. coccidiosis) can contribute to the development of the pathology, rather than in a different
in the liver presenting the classic necrotic lesions is nevertheless significant, as these are usually detected in UE in other animal species. The only reports of UE in laying hens refer to a focal necrotic duodenitis in which C. colinum has never been isolated but only identified
through molecular methods. A simultaneous C. colinum and C. perfringens co-infection in laying hens has never been reported. However, Beltran-Alcrudo et al. (2008) reported
isolation of both bacteria in a case of ulcerative enteritis in quails, considering C. colinum as primary agent of the pathology and attributing C. perfringens a secondary role. Following the diagnosis of intestinal clostridiosis the animals were treated for 5 days with tylosin in drinking water (50 g/100 L). It is well known that the strains of C. perfringens isolated from chicken are usually sensitive to tylosin but the lack of selective growth media
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for C. colinum makes the isolation from particularly contaminated organs difficult, as well as
Gholamiandehkordi et al., 2009). The fact that after treatment there was a remission in
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symptoms leads to the conclusion that C. colinum can also be susceptible to this molecule.
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the execution of tests to evaluate the susceptibility to antimicrobials (Martel et al., 2004;
References
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necrotic enteritis in broiler chickens: novel etiological consideration based on ultra-
Figure Legends
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Figure 1. Intestinal ulceration visible through the serous membrane of the intestine.
Figure 2a. Intestinal ulceration visible from mucosal surface. Note the extension of the
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pathologic process
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Figure 2b. Detail. Intestinal ulceration
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Figure 3. Ceca. Advanced ulcers filled with diphtheritic, necrotic debris.
Figure 4. Intestine. Uniform diffuse coagulation necrosis of the mucosa, diffuse
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lymphoplasmacytic infiltration and bacterial aggregates x20
Figure 5. Liver. Multifocal heterophilic-necrotic hepatitis associated with bacterial
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aggregates and diffuse steatosis of the hepatocytes X40
Table 1. Results of bacteriological and biomolecular examinations Outbreak
Subject
1 1a
Organ
Isolate
C.perfringens C. pefringens
PCR
Toxinotype
NetB gene
C. colinum
liver
C. colinum
-
-
positive
intestine
C.
A
positive
ntc
A
positive
ntc
2
intestine
C. perfringens
intestine
C.
2b
A
perfringens 4
intestine
C.
A
8
intestine
negative
positive
negative
positive
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perfringens C.
A
negative
positive
A
negative
positive
perfringens 9
intestine
C.
perfringens b
c
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June 2013; Dicember 2013; not texted
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a
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3
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perfringens
