Prevalence of Clostridium sPP. in diarrhoeic and healthy dogs ...

Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156Prevalence of Clostridium spp. in diarrhoeicand healthy dogsPrevalenza di Clostridium spp. in canidiarroici e saniPAROLE CHIAVE:Clostridium spp., cane, diarrea, feciKEY WORDS:Clostridium spp., diarrhoea, dog, fecesRiassuntoZerbini Laura, Ossiprandi Maria CristinaE’ stata effettuata una indagine epidemiologica allo scopo di verificare laprevalenza di Clostridium spp. nelle feci di cani diarroici e sani. Particolare attenzioneè stata rivolta all’isolamento di ceppi di C. perfringens e C. difficile, dal momentoche questi microrganismi sono stati associati nel cane a sindrome diarroica cronica eacuta di tipo emorragico.Il campione oggetto dello studio era composto da cani provenienti dalcanile municipale della città di Parma, da cani appartenenti a studenti e a personaledella Facoltà di Veterinaria di Parma, oltre che da cani di proprietà ricoverati pressol’Ospedale Didattico Veterinario.Globalmente, sono stati analizzati mediante esame colturale 95 campionifecali, raccolti nel periodo luglio 2006 - marzo 2007, di cui 36 appartenenti a canidiarroici e 59 a cani non diarroici. Sessantadue dei 95 campioni (65,3%) sono risultatipositivi per la presenza di 1 o più specie di Clostridium. Sono stati identificati 89differenti ceppi di Clostridium. In particolare, C. perfringens (15,7%) è risultatoessere la specie isolata più frequentemente, seguito da C. bifermentans (13,5%),C. clostridiiforme (13,5%), C. fallax (12,4%), C. beijerinckii/butyricum (11,2%),C. difficile (11,2%) e C. septicum (9,0%). Altre specie di Clostridium sono stateisolate in percentuali variabili dal 2,3% all’1,0%.Relativamente all’isolamento di ceppi di C. perfringens, 6 dei 14 campionidi feci risultati positivi appartenevano a cani diarroici (42,9%), mentre nel caso diC. difficile, l’80% dei campioni analizzati e risultati positivi all’esame colturale (8 di10) apparteneva a cani affetti da enterite.Ulteriori indagini, quale la rivelazione della produzione di tossine medianteSezione di Microbiologia ed Immunologia VeterinariaDipartimento di Salute Animale, Facoltà di Medicina Veterinaria - Università degli Studi di ParmaVia del Taglio n° 8 - 43100 Parma - Tel. 0521/032718mariacristina.ossiprandi@unipr.it143

Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156saggi ELISA e/o la ricerca delle relative sequenze geniche mediante reazioni diamplificazione genica, dovranno essere eseguite sui ceppi di C. perfringens e diC. difficile per poter valutare correttamente le implicazioni e il significatoepidemiologico di questi ritrovamenti nei campioni di feci di cani diarroici e non.AbstractThe purpose of this epidemiological study was to evaluate the prevalenceof Clostridium spp. among diarrhoeic and healthy dogs. Particular attention wasaddressed to C. perfringens and C. difficile isolation, since these microrganisms havebeen associated with acute and chronic large and small bowel diarrhoea, and acutehemorrhagic diarrhoeal syndrome in the dog.Some dogs were kennelled dogs, others belonged to students and staff ofthe Veterinary Faculty of Parma, and others were dogs admitted to the DidacticVeterinary Hospital.A total of 95 canine fecal samples (36 diarrhoeic and 59 non-diarrhoeic dogs),collected from July 2006 to March 2007, were analysed by culture assay. Sixty-two ofthe 95 fecal specimens (65.3%) were positive for one or more Clostridium spp. A totalof 89 different Clostridium strains were identified. The most common Clostridiumdetected was C. perfringens (15.7%), followed by C. bifermentans (13.5%),C. clostridiiforme (13.5%), C. fallax (12.4%), C. beijerinckii/butyricum (11.2%),C. difficile (11.2%), and C. septicum (9.0%). Other Clostridium species were isolatedin lower percentage, ranging from 2.3% to 1.0%.Relatively to C. perfringens isolation, 6 of the 14 positive fecal samples(42.9%) belonged to diarrhoeic dogs. In the case of C. difficile, the 80.0% (8 of 10) ofcanine fecal samples positive by culture assay belonged to dogs with enteritis.Further investigations (concerning, for example, the detection of toxinproduction by ELISA assays and/or the gene revelation by polymerase chain reactions)should be applied on C. perfringens and C. difficile strains to assess properly the fullimplications and the epidemiological meaning of these findings in fecal samples ofdiarrhoeic and non-diarrhoeic dogs.IntroductionThe development of microbiota of dogs begins at birth when the sterile foetusis colonized in the birth canal and by the immediate environment. The succession ofevents in bacterial colonization is similar in the human and canine intestine, with thevery first colonizers originating from the mother, followed by microbes benefitingfrom breast-feeeding and then drastically changing towards obligate anaerobes andheterogeneous flora as solid foods are introduced [10].The gastrointestinal tract of dogs is colonized by a variety of microrganisms.The number of microrganisms increases from approximately 10 2 -10 5 colony formingunits (CFU)/g in the proximal small intestine to 10 5 -10 9 CFU/g in the distal smallintestine, and then dramatically rises in the large intestine to approximately 10 10 -10 11 in the colon [1]. This quantitative proximal-distal gradient is accompanied144

Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156by complex qualitative changes from a predominantly aerobic flora in the smallintestine to a predominantly anaerobic flora in the large intestine. There is a widevariation in the bacterial flora between normal individuals, and concentrations maybe affected by a variety of circumstances or behaviours, including environment, diet,scavenging and coprophagia. Increased numbers of bacteria in the small intestinecarry an increased risk of a harmful outcome. Whether this is manifest clinically willdepend on individual circumstances, including the composition of the diet whichbacteria can convert to potentially harmful metabolites. The nature of a host responseto the increased load could also be important, and this may involve stimulation ofimmunoglobulin and mucus production, suppression of potentially damaging cellmediated immune responses, a compensatory production of structural and functionalepithelial cell proteins, and increased epithelial cell turnover. The threshold betweenapparent normality and clinical disease may therefore differ between individuals andbe influenced by a delicate balance between the microflora and the host. Clinicaldisease associated with bacterial overgrowth is a very common problem in dogs andoccurs when this balance is tipped against the host [1].Primary enteric bacterial pathogens can also cause acute clinical diseasesince they possess virulence factors with adverse effect on the intestine. Adherenceto the surface or invasion of the mucosa facilitate long-term colonization by certainenteropathogens, predisposing to a carrier status or chronic disease, the outcomeprobably depending on expression of virulence determinants and the host response[1].Since dogs are carnivorous, the total length of their intestine in relation to thebody length is somewhat shorter and the motility slower than in humans. As a wholethe digestive tract of dog, however, resembles that of humans and the physiologyis similar in many ways. Like humans, dogs utilize intestinal microbiota in theirphysiology and both are homothermic mammals. The pH values in the differentcompartments of the digestive tract are also comparable to those of human, being thepH in the dog stomach 3, in duodenum and jejunum 6, in ileum 7.5, in colon 6.5 andin feces 6.2 [10].The main cultivable bacterial groups and most common findings in humansand dogs are similar, including clostridia, bacteroides, streptococci, coliforms,enterococci, lactobacilli and veillonellae with increasing counts towards the largeintestine [10]. Most intestinal pathogens cause similar clinical symptoms in dogs asin humans (C. difficile, multiresistant Gram-negatives, enterococci, staphylococci).The most common intestinal dysfunction in dogs is diarrhoea. Another commondisturbance, small intestinal bacterial overgrowth, composing of Escherichia coli,enterococci and clostridia is associated with raised serum folate, reduced serumvitamin B12 concentrations and altered mucosal permeability and function [10].In the most cases of bacterial overgrowth, the cause cannot be identified, buthost factors known to predispose to bacterial overgrowth include defective gastric acidsecretion, interference with normal motility or stasis, and defective local immunity[1]. Other factors, as a potentially stressful or contamined environment, diet andcoprophagia, may also play a role and could contribute to the relatively high numbersof bacteria reported in kennelled dogs [1]. The damaging consequences of overgrowth145

Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156can involve competition by bacteria for calories and essential nutrients, productionof harmful metabolites, and direct damage to the intestinal mucosa interfering withintestinal function. Histological changes in intestinal biopsies of partial villus atrophyand lymphocyte/plasma cell infiltrate are only present in 30% of cases, and often themucosal damage cannot be seen by conventional light microscopy [1].Dogs with and without signs of intestinal disease can harbour most of theenteric infectious agents [5]. In the case of clostridial pathogens, particularly importantare C. perfringens and C. difficile. The clinical documentation of C. perfringensand C. difficile as causes of diarrhoea in dogs is clouded by the presence of thesemicrorganims as a normal component of the indigenous intestinal microflora [6].Clostridium perfringensClostridium perfringens is a Gram-positive, anaerobic, spore-formingbacillus. This microrganism may be one of the most widespread pathogen, inhabitingthe gastrointestinal tract of human beings and animals as well as terrestrial andmarine environments [7]. It has been associated with outbreaks of acute, often severediarrhoea in humans, horses, dogs and cats. The elaboration of four major toxins,alpha (α), beta (β), iota (ι), and epsilon (ε), is the basis for typing the microrganisminto five toxigenic phenotypes (A, B, C, D and E).Each type may also express a subset of at least 10 other established toxins,including C. perfringens enterotoxin (CPE), a well-characterized virulence factorwhose production is co-regulated with sporulation [6]. Although all five types canharbour the enterotoxin gene (cpe), studies have shown that the overall frequency ofenterotoxigenic strains is relatively low (∼5%), with most strains belonging to typeA. Virtually all strains isolated from dogs are type A, with only one published reportdocumenting a type C infection in five cases of canine peracute lethal hemorrhagicenteritis [7]. Although several studies have shown an association between theimmunodetection of CPE in fecal specimens and canine diarrhoea, the pathogenesisof C. perfringens-associated diarrhoea in the dog is not fully understood, because CPEis also detected in up to 14% of non-diarrhoeic dogs. Isolation of nonenterotoxigenictype A strains from a diarrhoeic specimen does not preclude involvement in disease,because there is a plethora of other virulence factors that have not been evaluated. Oneof these virulence factors is the recently characterized C. perfringens β2 toxin, whichhas been associated with both necrotic enteritis in piglets and equine typhlocolitis.Clostridium perfringens is a part of the normal canine intestinal flora andis readily cultured from more than 80% of diarrhoeic and non-diarrhoeic dogs.Diarrhoeal diseases associated with C. perfringens in the dog have primarily beenattributed to CPE, which has been shown to induce fluid accumulation and diarrhoeain a canine model when administered orally or directly in the intestinal lumen[7]. Dogs with C. perfringens-associated diarrhoea frequently exhibit large-boweldiarrhoea characterized by increased frequency of bowel movements with tenesmus,fecal mucus and hematochezia; however, clinical signs of enteritis or enterocolitisare also commonly seen [6]. Despite the fact that CPE is detected in up to 34% ofdiarrhoeic dogs, the fact that it is also detected in 5% to 14% of non-diarrhoeic dogs146

Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156makes it unclear as to whether or not CPE plays a primary or secondary role in thedevelopment of diarrhoea. There is currently no gold standard for the diagnosis ofcanine C. perfringens-associated diarrhoea. It has been well documented that cultureisolation of C. perfringens cannot be considered of diagnostic value for canineC. perfringens-associated diarrhoea. Culture may be useful in procuring isolates forthe application of molecular techniques like PCR for detection of specific toxin genesor molecular typing of isolated strains to establish clonality in suspected outbreaks.Two commercially available immunoassays are currently used in veterinarydiagnostic laboratories: a reverse passive latex agglutination assay (PET-RPLA,Oxoid, Basingstoke, Hampshire, England) and an enzyme-linked immunoassay(C. perfringens Enterotoxin Test, TechLab, Inc., Blacksburg, VA). It is important tonote that the performance characteristics for these assays have not been analyzed inthe dog, and there are concerns about their sensitivities and specificities. There havebeen no studies in the dog to date to determine whether C. perfringens-associateddiarrhoea is a result of ingestion of enterotoxigenic isolates or whether diarrhoea issecondary to disruption of the intestinal microenvironment, enabling proliferation orsporulation of commensal enterotoxigenic C. perfringens. Based on what is currentlyknown, however, it seems more likely that C. perfringens-associated diarrhoeainvolves commensal isolates [7].Clostridium difficileClostridium difficile is a Gram-positive, anaerobic, spore-forming bacilluswhich is the major cause of antibiotic-associated pseudomembranous colitis inhuman patients. It has also been associated with diarrhoea and enterocolitis in foalsand adults horses, as well as diarrhoea in dogs [6].Three toxins produced by C. difficile have been described: toxin A (anenterotoxin), toxin B (a cytotoxin), and C. difficile adenosine diphosphate (ADP)-ribosyltransferase (CDT). Diseases associated with C. difficile have primarily beenattributed to the activity of toxins A and B, and strains have historically been thoughtto produce both toxins (toxigenic isolates) or neither (nontoxigenic). There areincreasing reports of variant strains isolated from human clinical cases of C. difficileassociateddisease (CDAD) that produce only toxin A or toxin B, however [7].Current diagnosis of C. difficile-associated diarrhoea is primarilybased upon detection of toxin A and/or toxin B in fecal specimensby ELISA. Isolation of the microrganism alone is not sufficient fordiagnosis, due to the presence of nontoxigenic strains. In addition,previous studies have reported no significant difference in the isolation ofC. difficile from diarrhoeic and non-diarrhoeic dogs [6]. Toxigenic C. difficile has beenisolated from dogs with chronic diarrhoea, and reports have documented a variablecarriage rate of C. difficile ranging from 0-40% in diarrhoeic and non-diarrhoeic dogs[3]. Despite the lack of a significant difference in the isolation rates of C. difficile indiarrhoeic and non-diarrhoeic dogs, an association has been documented betweenthe presence of diarrhoea and detection of toxin A or toxin B, or both, in canine fecalspecimens [3].147

Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156Antibiotic administration, especially in hospital environments, is the mostcommonly reported as predisposing factor for the development of CDAD in peopleand horses, although there have been reports of CDAD in the absence of antibioticadministration. In contrast, administration of antibiotics does not seem to predisposedogs to CDAD [7].Toxigenic C. difficile can be isolated from up to 94% of neonate dogs in theabsence of clinical signs of disease. Clinical signs that have been associated withcanine C. difficile infection range from asymptomatic carriage to a potentially fatalacute hemorrhagic diarrhoeal syndrome. As with C. perfringens, there does not seemto be a specific anatomic localization of clinical signs, and recent studies have shownthat dogs with suspected C. difficile-associated diarrhoea commonly have signsof small and large intestinal diarrhoea as well as diffuse disease characterized byconcurrent involvement of both the small and large intestine [7].As with C. perfringens, isolation of C. difficile from diarrhoeic specimensis of little diagnostic value, except for procuring strains for detection of toxin genesand typing. Limitations in the isolation of C. difficile in dogs are underscored by thefindings of several studies demonstrating no significant difference in isolation rates(0-40%) between non-diarrhoeic and diarrhoeic dogs, as above mentioned.Diagnosis of C. difficile-associated diarrhoea traditionally has been basedon positive fecal assays for toxin A or toxin B. The current gold standard assay isthe cell culture cytotoxicity assay, which detects toxin B activity; however, thisassay is expensive and requires up to 48 hours for conformation of a negative result.There are currently several commercially available immunoassays for detection ofC. difficile toxin A or for detection of both toxins A and B in fecal samples (for example,Premier Toxins A & B, Meridian Bioscience, Inc, Cincinnati, OH; C. difficile TOXA/B II, TechLab, Inc., Blacksburg, VA; ProSpecT Clostridium difficile Toxin A/B,Remel, USA). Because of increasing reports of toxin A-negative and toxin B-positivestrains isolated from clinical cases, the use of ELISA kits that detect both toxins isgaining preference. None of the commercial ELISA kits currently available havebeen validated in the dog, however, and there is no standard assay used by veterinarydiagnostic laboratories. This fact is concerning, given the wide range of specificities(66-100%) and sensitivities (33-95%) that have been reported for many of the kitswhen analyzed for human fecal toxin detection.Detection of toxigenic C. difficile strains after culture has been shown to be oflittle diagnostic value in the dog. First, there is no significant difference in detection oftoxigenic strains between diarrhoeic and non-diarrhoeic dogs, and, second, isolationof the microrganism is difficult, costly, and time-consuming. Nevertheless, studies inhuman beings have begun bypassing culture of the microrganism and looking for thepresence of specific toxin genes in DNA obtained from fecal specimens. Sensitivitiesof PCR detection methods have been reported to be between 96% and 100% whencompared with the cytotoxicity assay [7].The aim of this study was to define the prevalence of Clostridium spp. in95 fecal samples (36 diarrhoeic and 59 non-diarrhoeic) collected from dogs at theVeterinary Faculty of Parma. Especially, we payed attention to C. perfringens andC. difficile isolation.148

Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156Materials and methodsFecal samples were collected from 95 diarrhoeic and non-diarrhoeic dogs,41 female and 54 male, over an 8-mo period (20 th July 2006 - 20 th March 2007). Ageof the diarrhoeic dogs (n= 36) ranged between 6 months and 12 years (mean, 4.2 yr;median, 3 yr), and that of the non-diarrhoeic dogs (n= 59) ranged between 3 monthsand 12 years (mean, 3.8 yr; median, 3 yr).Some dogs (n= 38) were kennelled dogs; others belonged to veterinarystudents and staff of the Veterinary Faculty of Parma (n= 47). Ten dogs were admittedto the Didactic Veterinary Hospital.All fecal specimens were naturally voided. Assays were performed on fecescollected within 3 hours. After analysis, samples were immediately stored at -20°C.All fecal samples were cultured onto prereduced blood agar plates,containing vitamine K, sheep blood and haemin (Schaedler agar, Oxoid, Basingstoke,Hampshire, England), and at the same time inoculated in Cooked Meat Broth (Oxoid,England). Samples were also streaked onto prereduced selective medium containingcycloserine-cefoxitin-fructose agar (CCFA) for the isolation of C. difficile. Plateswere incubated anaerobically at 37°C for 48-72 hours. After 72 hours of anaerobicincubation in Cooked Meat Broth, the samples were subjected to heat shock forspore selection and then cultured onto Schaedler agar and/or CCFA to enhance sporegermination and increase culture yield.For C. perfringens identification, colonies showing characteristic dualhaemolytic zones (Figure 1) were picked up and identified by Gram’s stain andbiochemical tests utilizing Rapid ID 32A (bioMérieux SA, Marcy-l’Etoile, France).Preliminary identification of C. difficile was based on colonial appearance (Figure2), odor (horse manure), lack of aerotolerance, cell morphology after Gram stainingand typical chartreuse fluorescence under ultraviolet light. Final identification wasperformed by using a rapid latex slide agglutination test (C. difficile, Oxoid, England)and Rapid ID 32A biochemical profiles (bioMérieux SA, France). The other clostridiawere identified only biochemically (Rapid ID 32A, bioMérieux).All isolates were stored on cryopreservation beads (MAST Diagnostics,D.I.D, Diagnostic International Distribution S.p.A., Italy) at -70°C.Figure 1: Typical colonies of Clostridium perfringens onto Schaedler agar showingcharacteristic dual haemolytic zones149

Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156Figure 2: Typical colonies of Clostridium difficile that could be greyish or whitishcolour, opaque or lucid, flat, circular or occasionally rhizoid and non-haemolyticResultsSixty-two fecal samples were positive for Clostridium spp. presence(62/95 samples, 65.3%). The other 33 samples were negative for Clostridium spp.Eigthy-nine Clostridium spp. were isolated and identified from the 62 positive fecalspecimens. The most common Clostridium detected was Clostridium perfringens(14/89 strains, 15.7%), followed by C. bifermentans (12/89, 13.5%), C. clostridiiforme(12/89, 13.5%), C. fallax (11/89, 12.4%), C. beijerinckii/butyricum (10/89, 11.2%),C. difficile (10/89, 11.2%), and C. septicum (8/89, 9.0%). Other clostridia isolatedwere: 2 strains of C. paraputrificum, 2 of C. glycolicum and 2 of C. tyrobutyricum(2/89, 2.3%), 1 strain of C. tetani, 1 of C. botulinum, 1 of C. innocuum, 1 of C. tertium, 1of C. sporogenes and 1 of C. histolyticum (1/89, 1.1%). Different species of clostridiawere isolated in the same fecal sample. In Table 1, the prevalence of Clostridium spp.in fecal samples of diarrhoeic and non-diarrhoeic dogs is summarized.Concerning C. perfringens, 6 of the 14 positive fecal samples (42.9%)belonged to dogs with enteritis. In one case, the dog, affected by megaesophagus,was subjected to antibiotic therapy for enteritis and C. difficile together withC. perfringens were also isolated. The other 8 samples (57.1%) belonged to healthydogs (Table 2).In the case of C. difficile isolation, 8 of the 10 canine fecal samples (80.0%)belonged to diarrhoeic dogs, of whom 4 were subjected to antibiotic treatment and theenteritis followed the therapy. From one dog (megaesophagus) treated for enteritis,C. perfringens together with C. difficile were isolated. Three dogs were not treated.Another dog, admitted to the Didactic Veterinary Hospital, was subjected to antibiotictreatment but was non-diarrhoeic. One dog was an healthy dog (Table 3).150

Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156Table 1: Prevalence of Clostridium spp. in 95 fecal samples of diarrhoeic andhealthy dogs in the period 20 th July 2006 - 20 th March 2007No. fecal %samplespositiveClostridium perfringens 10 16.1Clostridium bifermentans 6 9.7Clostridium fallax 6 9.7Clostridium septicum 4 6.44Clostridium clostridiiforme 3 4.83Clostridium difficile 2 3.22Clostridium beijerinckii/butyricum 1 1.61Clostridium paraputrificum 1 1.61Clostridium glycolicum 1 1.61Clostridium tyrobutyricum 1 1.61Clostridium tetani 1 1.61Clostridium botulinum 1 1.61Clostridium histolyticum 1 1.61Clostridium beijerinckii/butyricum Clostridium clostridiiforme 3 4.83Clostridium difficile Clostridium bifermentans 3 4.83Clostridium perfringens Clostridium beijerinckii/butyricum 2 3.22Clostridium difficile Clostridium perfringens 1 1.61Clostridium tertium Clostridium septicum 1 1.61Clostridium septicum Clostridium beijerinckii/butyricum 1 1.61Clostridium tyrobutyricum Clostridium innocuum 1 1.61Clostridium clostridiiforme Clostridium fallax 1 1.61Clostridium glycolicum Clostridium septicum 1 1.61Clostridium fallax Clostridium bifermentans 1 1.61Clostridium clostridiiforme Clostridium paraputrificum 1 1.61Clostridium difficile Clostridium beijerinckii/butyricum 1 1.61Clostridium fallax Clostridium septicum 1 1.61Clostridium difficile Clostridium bifermentans Clostridium 1 1.61clostridiiformeClostridium difficile Clostridium sporogenes 1 1.61Clostridium bifermentans Clostridium fallax Clostridium 1 1.61beijerinckii/butyricumClostridium beijerinckii/butyricum Clostridium clostridiiforme 1 1.61Clostridium fallaxClostridium perfringens Clostridium clostridiiforme 1 1.61Clostridium difficile Clostridium clostridiiforme 1 1.61Total 62151

Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156Table 2: Prevalence of Clostridium perfringens strains isolated in fecal samples ofdiarrhoeic and healthy dogsTable 3: Prevalence of Clostridium difficile strains isolated in fecal samples ofdiarrhoeic and healthy dogsDiscussionIn this epidemiological study we assessed the prevalence of Clostridium spp.in 95 canine diarrhoeic and non-diarrhoeic fecal samples. Except for C. perfringensand C. difficile, there are not important data in literature for the other Clostridium spp.On the contrary, C. perfringens and C. difficile are common causes of enteritis andenterotoxemias in both domestic animals and humans. These microrganisms havebeen associated with acute and chronic large and small bowel diarrhoea, and acutehemorrhagic diarrhoeal syndrome in the dog [8].In our study, Clostridium perfringens was commonly cultured from fecesof dogs (15.7%). Percentages of positive cultures were enough similar in healthydogs and in diarrhoeic dogs (8 of 14, 57.1%, versus 6 of 14, 42.9%, respectively).This difference is not significant (P= 0.571, Chi-square test). Since we didn’t assessthe enterotoxin (CPE) production from C. perfringens isolates, we cannot correlatethe C. perfringens isolation with clinical disease in dogs, also considering that thismicrorganism may be part of the normal flora [6]. Actually, current diagnosis ofC. perfringens-associated diarrhoea in dogs and cats is made on detection of CPE infecal samples in conjunction with clinical signs of disease [6].Relatively to C. difficile, the isolation rates of this microrganism fromdiarrhoeic dogs (8/10, 80.0%) and non-diarrhoeic dogs (2/10, 20.0%) were statistically152

Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156different (P= 0.023, Fisher’s test). This is in disagreement with previous reports inwhich significant differences were not found in the isolation rates of C. difficilebetween the 2 groups [3]. Four of ten C. difficile-positive dogs expressed enteritisafter antibiotic therapy. Probably, antibiotic administration caused the overgrowthof C. difficile in intestine of the dogs, predisposing the animals to enteritis. Infact, a previous study has reported that the carriage rate of C. difficile and toxinsin animals including dogs that had recently received antibiotics was higher thanin the nonantibiotic-treated group, although these differences were not statisticallysignificant [3]. Another study has indicated that the presence of C. difficile in caninefeces was evident only after administration of antibiotic [9]. However, results ofseveral studies have supported the notion that antimicrobial treatment is not requiredfor C. difficile-associated disease (CDAD) development in dogs [3].Since we did not research the toxins A and B (neither in vivo directly in fecesnor in vitro from the C. difficile isolates), which seem the primary virulence factorsinvolved in the pathogenesis of CDAD, we cannot correlate the C. difficile isolationwith dog illness. However, we performed on the 10 C. difficile isolates a duplexPCR for the revelation of tcdA and tcdB genes, encoding for toxin A and toxin B,respectively [11]. The majority of C. difficile strains (6/10, 60.0%) were toxigenic(tcdA+/tcdB+) on the basis of the results of PCR assay (data not shown). The carriagerates of toxigenic isolates in diarrhoeic dogs (5/6, 83.3%) were higher than thosein non-diarrhoeic dogs (1/6, 16.7%). However, this difference was not statisticallysignificant (P= 0.08, Fisher’s test).It could be interesting to verify if tcdA/tcdB-positive strains by PCR willbe able to produce the two toxins also in vitro, because the presence of genes is notalways correlated with their expression.ConclusionsIt is particularly important to seek C. perfringens and C. difficile presencein canine fecal samples. However, C. perfringens-associated diarrhoea in the dogseems to be a complex disease, the pathogenesis of which is still poorly understood.Furthermore, despite various reports that have documented an association betweenthe detection of CPE and the presence of diarrhoea, it is still unclear whether or notC. perfringens is a primary cause of canine diarrhoea or a secondary determinant.Much work still needs to be done to clarify the exact role of C. perfringens incanine diarrhoea. Based on the results of several studies, the optimum diagnosticapproach for canine C. perfringens-associated diarrhoea is the use of a CPE ELISAin conjunction with PCR for detection of enterotoxigenic strains procured aftera heat shock or alcohol shock treatment [7]. To date, different PCRs (duplex andmultiplex) were evaluated for the revelation of genes encoding for C. perfringenstoxins (CPE, toxin α, toxin β, toxin β2, toxin ι and toxin ε) only for research purposes[2, 4]. It will be useful to evaluate these methods on our isolates for the presence ofC. perfringens toxin genes, especially of the CPE gene. The production in vivo and/orin vitro of CPE should be also investigated to correlate the C. perfringens isolationwith diarrhoea in dogs.153

Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156C. difficile is a significant enteropathogen in human beings and horses, whereinfection is most commonly associated with disruption of the normal microflora,followed by colonization by toxigenic strains. The role that this microrganism playsin dogs is not well defined, and only a few studies evaluating the presence of toxinsin diarrhoeic and non-diarrhoeic animals have been done [7].There is no consensus between veterinary diagnostic laboratories regardingdiagnosis of C. difficile-associated diarrhoea in the dog. The apparently highprevalence of ELISA-positive, culture-negative canine specimens obtained withsome assays is questionable, considering that these commercial assays have neverbeen validated in the dog; therefore, such data may represent the consequence offalse-positive results [7].Ideally, the application of PCR assays directly on diarrhoeic fecal specimensand/or on C. difficile isolated strains for the detection of toxin A and B genes combinedwith ELISA tests for the demonstration of toxin production (in vivo and in vitro)should be implemented for diagnosing canine CDAD.AcknowledgementsThe authors wish to thank Mrs Cinzia Reverberi and Mr. Roberto Lurisi for theirtechnical support.RingraziamentiSi ringrazia la sig.ra Cinzia Reverberi ed il sig. Roberto Lurisi per l’assistenza tecnicaprestata.References1. Batt R.M., Rutgers C. (1997) Bacteria and intestinal disease in dogs. GDBATechnical Review no 11. UK.2. Baums C.G., Shotte U., Amtsberg G., Goethe R. (2004) Diagnostic multiplexPCR for toxin genotyping of Clostridium perfringens isolates. Vet. Microbiol.,100: 11-16.3. Chouicha N., Marks S.L. (2006) Evaluation of five enzyme immunoassayscompared with the cytotoxicity assay for diagnosis of Clostridium difficileassociateddiarrhea in dogs. J. Vet. Diagn. Invest., 18: 182-188.4. Fach P., Popoff M.R. (1997) Detection of enterotoxigenic Clostridium perfringensin food and fecal samples with a duplex PCR and the slide latex agglutinationtest. Appl. Environ. Microbiol., 63: 4232-4236.5. Hackett T., Lappin M.R. (2003) Prevalence of enteric pathogens in dogs ofNorth-Central Colorado. J. Am. Anim. Hosp. Assoc., 39: 52-56.6. Marks S.L. (2003) Bacterial gastroenteritis in dogs and cats. In Proceedingsof 28 th World Congress of the World Small Animal Veterinary Association.Bangkok, Thailand, October 24-27, 2003.7. Marks S.L., Kather E.J. (2003) Bacterial-associated diarrhea in the dog: a criticalappraisal. Vet. Clin. Small Anim., 33: 1029-1060.8. Marks S.L., Kather E.J. (2003) Antimicrobial susceptibilities of canine154

Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156Clostridium difficile and Clostridium perfringens isolates to commonly utilizedantimicrobial drugs. Vet. Microbiol., 94: 39-45.9. Martirossian G., Sokol-Leszczynska B., Mierzejewski J., Meisel-Mikolajczyk F.(1992) Occurence of Clostridium difficile in the digestive system of dogs. Med.Dosw. Mikrobiol., 44: 49-54.10. Mentula S. (2005) Analysis of canine small intestinal and fecal microbiota.Prevention of ampicillin-induced changes with oral β-lactamase. AcademicDissertation. Publication of the National Public Health Institute, KTL A8, June2005, 1-85.11. Spigaglia P., Mastrantonio P. (2002) Molecular analysis of the pathogenicitylocus and polymorphism in the putative negative regulator of toxin production(TcdC) among Clostridium difficile clinical isolates. J. Clin. Microbiol., 40:3470-3475.155

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