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Clostridial Enterocolitis
EMERGINC INFECTIOUS DISEASES
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CLOSTRIDIAL ENTEROCOLITIS Robert L. Jones, DVM, PhD
Acute enterocolitis and diarrhea associated with clostridia are garnering greater recognition as important clinical problems. An e lectronic search for references on equine clostridial entemcolitis illustrates the increasing significance of the disease in the 19905. This is evidenced by 9 citations in the 1970s, 19 in the 1980s, and nearly 50 in the 1990s that deal with epidemiology, virulence factors, improved diagnostic tests, and evaluation of treatment outcomes. Based on these studies, clostridia have been firmly incriminated as playing a major causative role in enterocolitis in horses. Clostridium pClji'illgCIIS is associated with diarrhea in foals and enterocolitis in horses of all ages. C. difficile has eme rged as an important cause of nosocomial and antibiotic-associated enterocolitis in horses. Included among the reasons that may account for the emergent role of clostridia in enterocolitis and diarrhe,l are an increased awareness among owners and veterinarians resulting in more detailed and comprehensive investigations, increased sensitivity of diagnostic assays, changing management conditions that modify natural resistance to this syndrome, and the impact of antimicrobial use and resistance on the initiation of novel clinical problems. The genus ClostridiunI includes many species of obligatory anaerobic to aerotolerant spore-forming gram-positive rods. Because sporulation increases resistance to drying, heat, irradiation, and chemicals, clostridia are generally ubiquitous in nature, existing principally in soil and the intestinal tracts of many animals. C. peljrillgclI s and C. difficile are the species most frequently isolated from cases of equine enterocolitis. C. scptiCllIII," C. sordelli," C. Cildaveris,'9. '" C. paraputrificulII,5I1 and at least six unidentified clostridia'" have been recovered from acute enterocolitis in the horse. In addition, C. spirojormc,"' C. botulinulIl types C and D producing C2 toxin,''' and C. butyricwn'" have bee n associated with acute enterocolitis in other species. Although exogenous spread from animal to animal has been confirmed during outbreaks of disease, endogenous infections involving clostridia that a re a part of the host's own microflora are probably much more common. The basic biology of clostridia and the clinical
From the Depa rtment of Microbiology, College of Vete rinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado
VETERINARY CLINICS OF NORTH AMERICA: EQUINE PRACTICE VOLUME 16· NUMBER 3 · DECEMBER 21100
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management of enterocolitis have been described in detail.lJ, 33,10, 4", 52 In this article, emphasis is placed on recent advances in our understanding of the disease, including the discovery of C. perfringens (32 toxin, the epidemiology of C. perfringens, nosocomial and antibiotic-related enterocolitis associated with C. difficile, and the effect of factors such as antibiotic use and hospitalization on the occurrence of equine enterocolitis.
CLOSTRIDIUM PERFRINGENS C. perfringens, also called C. welchii, is phenotypically and genetically highly variable; this provides for differences in virulence among strains and in their ability to cause disease. C. perfringens is a prolific toxin producer that is capable of expressing as many as 17 exotoxins, although individual strains produce only a defined subset of these toxins. 1, 37, 4",52 This characteristic forms the basis for a toxin-typing system for classifying C. perfringells isolates into five types (A, B, e, D, and E) based on an isolate's ability to produce one or more of the "major lethal" toxins. Other varieties or subtypes have been described within or in addition to these types based on the production of apparently less important toxins. Other typing systems are based on a strain's ability to produce enterotoxin, antigenic relatedness based on cell wall antigens (serotypes), and molecular types or genotypes based on nucleic acid analysis.
Prevalence of Clostridium perfringens C. perfringens is widely distributed as vegetative cells and spores in soil and fecal matter, which ensures that the organism is frequently present on surfaces exposed to dust contamination, including many feed items.' C. perfringens is one of the first organisms to colonize the neonatal intestinal tract, and a proportion of any healthy population of mammals can be expected to carry the organism." Most reports probably underestimate the true prevalence of C. perfringens in the intestinal tract of horses and do not differentiate between organisms in transit through the tract and those that are permanently established as part of the indigenous microflora. Geographic and seasonal variations in environmental concentrations of C. perfringens undoubtedly alter the quantity and shedding of this bacterium. Furthermore, differences in sampling and detection methods may explain the disparity in prevalence that has been reported in different studies. C. perfringens may exist in one or more states; these include the vegetative cell, the sporulating cell, the endospore, and the germinating endospore, each of which requires unique culture conditions to optimize the chances of recovery.41 When detected in the feces of healthy horses, C. perfrillgens is usually isolated only in small numbers. A count of less than 103 colony forming units (cfu) per gram of feces is considered normal,n, 33 with 75% of positive fecal cultures yielding less than 100 cfu / g." Using relatively insensitive direct plating methods, C. perfrillgens was detected in feces from 2 of 32 (6'Yo) healthy foals" and 12 of 50 (24%) horses. 53 When using a combination of direct plating, enrichment, and spore selection methods, C. perfringens was isolated from the feces of 4 of 29 (14%) healthy foals, 32 23 of 124 (19%) foals," 60 of 223 (27%) foals,.3 173 of 271 (64%) foals,"1 and 77 of 105 (73%) healthy horses."
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Association of Clostridium perfringens with Enterocolitis
The presence of C. perfringens in large numbers in feces and intestinal contents has been associated with acute equine enterocolitis in Europe 33 and North America.'"' 52 C. perfringells was isolated from cases of diarrhea from 12 of 304 (4%) foals/ and 58 of 365 (16%) foals," and from 57% of 421 foals and 27% of 223 healthy foals. 41 It was associated with 68'1'0 of 22 foals with diarrhea that died,4344 and'with 47 of 54 foals with rapidly progressive enterocolitis that often had a fatal outcome, despite intensive supportive therapy." C. perfringens was isolated from 95°;\, of samples obtained from horses with anterior enteritis compared with 60')(, of samples from other types of COlic. 21 Subtyping the 165-235 intergenic spacer revealed a new genotype of C. perfringells that seems to playa major pathogenic role in foal diarrhea. It was isolated from 12 of 14 fatal cases, but the toxin type was not identified." Toxin Types of Clostridium perfringens
The major toxin produced by C. perfringells type A is a toxin; this is also produced in varying amounts by all typeable strains but not consistently in lethal quantities by some type A strains. The a toxin has both phospholipase C and sphingomyelinase activities and is able to elicit a variety of subtle effects on eukaryotic cell metabolism, including activation of the arachidonic acid cascade and stimulation of protein kinase C activity.211 There is direct evidence for a toxin activity in toxic infections of tissue 211 Although type A is the predominant C. perfrillgells type isolated from healthy horses, II, 21 foals with diarrhea, 11,12,41 or foals and adult horses with enterocolitis,'J.2! there is little direct evidence incriminating this toxin as a significant enteric virulence factor." A variant of a toxin that is more resistant to intestinal chymotrypsin than the toxin from nongastrointestinal disease isolates has been identified/" suggesting that it may have some role in enterocolitis. The presence of hemorrhagic necrotizing lesions in some foals 9 from which type A is isolated suggests that unidentified factors other than a toxin or enterotoxin are contributing to the pathogenesis of the intestinal lesions. A novel toxin produced by C. perfrillgells and recently designated as (32 toxin may play an important role in the pathogenesis of necrotizing intestinal disorders in horses.!9 25 Both (32 and (3 toxins have comparable biologic activities, with each capable of causing hemorrhage and necrosis of the intestinal wall, lethal infection in mice, and cytotoxicity in selected cell cultures. The (32 and 13 toxins lack significant amino acid identity; this results in limited antigenic crossreactivity. The (32 toxin gene was found in 15 isolates of C. perfrillgells recovered in high numbers (>10" cfu I g) from the intestinal contents of horses that died with enterocolitis. I'> In a second survey,25 an undesignated toxin type of C. perfringens containing the a and 132 toxin genes was found in 75'X, of samples of intestinal contents and biopsy specimens of the intestinal wall from horses with acute enterocolitis. It was found in a lower percentage (62%) of horses with other intestinal disorders but was not recovered at all from the feces of 58 control horses. Although currently unproven, the newly identified (32 toxin, which was present in isolates from horses that would otherwise have been classified as type A, may play an important role as a cytotoxin in the pathogenesis of toxic intestinal disorders in horses. Some C. perfi'illgells strains produce enterotoxin, a virulence factor best known as a cause of food poisoning in human beings. Enterotoxin causes
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cell membrane pore formation, followed by altered permeability, inhibition of macromolecular synthesis, cytoskeletal disintegration, and lysis of cells. Only 2% to 6% of the global C. perfringells population carries the enterotoxin gene, and these isolates are almost always type A.37 Enterotoxigenic strains of C. perfringens were found in feces of 7 of 50 (14'Yo) horses," and in 13% of horses l5 in general surveys. In one study, enterotoxigenic strains were found in 16'X) of horses with diarrhea but not in age- and seasonally matched controls. I? In cases of foal diarrhea, enterotoxigenicity was no more common among isolates than in matched controls. The prevalence of enterotoxin-positive strains of C. perfringens as determined by polymerase chain reaction (PCR) assay was too low (9.7%) for them to be considered a pathogenic subtype. II . '2 Several authors have discounted the importance of enterotoxin in the pathogenesis of C. pelfrillgensassociated foal diarrhea, II. 17.33, Sil although others consider that it may still have a minor role to play in cases of foal diarrhea that resolve but not in those that have a fatal outcome. 42 C. perfringens types B, C, and D have been associated with severe necrotizing, hemorrhagic enterocolitis in foals. 4K , ,2 Type C is the most commonly reported clostridial enteric pathogen in foals in North America. IK,", The [) toxin of types Band C induces inflammation and progressive necrosis of intestinal mucosa with epithelial cell death and desquamation, followed by further bacterial invasion, multiplication, and even more toxin production!' Lesions found in the jejunum and ileum usually consist of acute hemorrhagic enteritis with necrosis of villi, with large numbers of gram-positive rods demonstrable in stained smears and sections. If diarrhea is present, it usually occurs for only a brief period before death. The precise biologic activity of the E toxin of C. perfringens type D, which increases intestinal permeability and causes toxicity of the central nervous system, has not been identified. There is limited evidence that this toxin type can be a significant pathogen in horses.
CLOSTRIDIUM DIFFICILE C. difficile is currently the most frequently identified cause of nosocomial and antibiotic-associated diarrhea in human beings, even though it can only be isolated and identified in approximately 25% of cases! The pathogenesis of C. difficile infection in humans has been the subject of several extensive reviews. I, " 5,17,22,27,36,4" C. difficile produces several hydrolytic enzymes and at least five toxic factors.' Only toxin A, which is primarily an enterotoxin, and toxin B, a cytotoxin, have been studied in sufficient detail to confirm their involvement in enterocolitis. s Ingested spores survive the low pH of the stomach and upper small intestine, germinate in the terminal ileum, and multiply in the colonic lumen. In the absence of competition by the indigenous microflora (colonization resistance), C. difficile may grow to high numbers (>10" cfu / g). The critical time in terms of susceptibility to infection is the period just after reduction of the colonic flora and before they are re-established." As C. difficile increases in number, it produces toxins and enzymes that damage tissue. When these toxins are produced, they act synergistically to induce damage to intestinal tissue and disrupt cell-to-cell tight junctions, resulting in acute inflammation, mucosal permeability, fluid accumulation, and necrosis of the epithelium. s 23 Released nutrients, and possibly locally induced anoxia, may, in turn, stimulate C. difficile growth and toxin production. Ultimately, this can lead to cell detachment, local necrosis, hemorrhage, and pseudomembranous lesions. In humans, the result of
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C. difficile infection can range from asymptomatic colonization to severe diarrhea, pseudomembranous colitis, toxic megacolon, colonic perforation, and death." 22, 27 Epidemiology of Clostridium difficile Infections
The heat- and acid-resistant spores of C. difficile are capable of persisting for months to years in the environment.' " C. difficile has been found inconsistently in the environment, suggesting a patchy environmental distribution or more probably a reflection of variable sampling methodologies and insensitive culture methods. 7 In South Wales, C. ditJicile was found in 7'1,) of samples from water (river, sea, and lake) and soil, 1'Yo of horse feces, and 17°;\, of environmental samples from veterinary clinics.· 7 In humans, the consensus is that 1% to 2'Yo (range, 0%-15%) of adults and up to 60% of healthy neonates carry C. difficile.'6 Animal reservoirs have been recognized, and a great variety of wild, farm, and domestic species may carry the organism. 7 It is not known if intestinal carriage is a temporary or permanent state in animals and humans. Carriage of C. difficile, even in large numbers, is not associated with increased risk of disease. Although person-to-person spread has been considered the likely source of infection for most documented outbreaks of C. difficile enteritis in hospitals, the environment and endogenous carriers are increasingly being suspected as sources of infection and reinfection'" for some cases of diarrheal disease in the general community." In 1987, toxigenic C. difficile was first implicated as a cause of equine enterocolitis in a group of foals with diarrhea'4 and in a series of foals with hemorrhagic necrotizing enteritis. 3 Within the past decade, numerous studies have provided evidence of the causative role of C. difficile in nosocomial diarrhea and acute colitis' 3,1-1, , . " 36, .h, 5 1. 3·) as well as in antibiotic-associated diarrhea and enterocolitis of adult horses,",3. 1.,2 1 .5 C. difficile is infrequently isolated from clinically normal adult horses and foals. There is some suggestion that it might be more prevalent in horses than isolation rates would indicate. 22 When identified, the prevalence is usually less than 2%2,3,24,,. ,6. 43 It is quite likely that the prevalence of C. difficile carriers would be much greater if more sensitive culture methods or molecular detection methods such as PCR assay were used, however. The prevalence and risk factors for C. difficile infection in foals have not been established. Some foals develop diarrhea without a history of antimicrobial treatment/" whereas others become asymptomatic carriers and potential reservoirs of C. difficilc after antimicrobial treatment.' Other factors that require evaluation in connection with C. difficilc infection include intercurrent rota virus infection"l and lactose intolerance. s• In 1993, C. ditJicile was first associated with enterocolitis in an adult horse.·' Since then, C. difficile has been associated with major outbreaks of nosocomial enterocolitis in horses in a number of equine hospitals in Europe 3, 1.,51 and the United States,'"' '6 leading one author to suggest that most hospitals experience sporadic episodes of the disease.·" Prior antimicrobial use (systemic, oral, or both) was a common factor in the history of most of these cases. 2• 14, 34-3<> The importance of antibiotic use as a risk factor was supported by findings from Sweden, where C. difficile or its cytotoxin was identified in fecal samples from 5 of 11 mares that developed acute colitis when their foals were treated orally with erythromycin and rifampicin for Rhodococcus equi infection. 3 C. difficile or its cytotoxin has been identified in the feces and intestinal contents of adult horses with acute colitis or diarrhea in several other studies. 16 - IH, 25, .X The nature of the interactions between C. difficilc and hospitalization, antimicrobial treat(1
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ment, and preexisting gastrointestinal disease have not been determined. C.
difficile has been associated with acute colitis and diarrhea in adult horses sufficiently frequently, however, for several authors to recommend routine testing for C. difficile and its cytotoxin in any horse that presents with acute colitis and diarrhea after treatment with antibiotics or in association with hospitalization.I. 3. 16
CLINICAL FEATURES
Clostridial enterocolitis occurs in horses worldwide, ranging in severity from asymptomatic to acute fulminating enterocolitis with a high case fatality rate. It usually occurs on a sporadic basis; however, occasionally, there can be clusters of cases in foals on breeding farms and nosocomial outbreaks. There seem to be two major manifestations of clostridial enterocolitis in foals: diarrhea with mild enteritis and severe hemorrhagic necrotizing enterocolitis. In respect to the former, diarrhea is the primary clinical sign, and whereas supportive treatment can be beneficial, many foals make spontaneous recoveries."· 43 Because of this, the incidence of this form of clostridial disease is probably underreported. The more severe form of clostridial enterocolitis presents as a rapidly progressive disease that often has a fatal outcome, despite the implementation of intensive care measures. Foals typically present with diarrhea (approximately 50% may have hemorrhagic diarrhea), obtunded mentation, clinical dehydration, colic, and tachypnea. '8 Acute enterocolitis in adult horses varies from moderate to severe, with diarrhea ranging from loose pasty to watery hemorrhagic feces, accompanied by abdominal pain, fever, d ecreased appetite, septic shock, and even sudden death.l2· 33 The onset of clinical signs is usually rapid but may extend over a period of several days in some cases. Evidence of dehydration and shock is evident on postmortem examination. The intestines may be distended with fluid, and discoloration of the serosa is frequently present. Necrosis of the mucosa may range from superficial to deep and may be accompanied by mild mucosal edema to a severe fibrinous hemorrhagic exudate on the mucosal surface. EPIDEMIOLOGY
Many horses are temporary or permanen t enteric carriers of clostridia. Some of these animals are susceptible to disease, although others seem to remain resistant to disease for extended periods, during which they remain culturepositive for these organisms. A number of risk factors are thought to be involved in equine clostridial enteritis. These include stressful events such as racing, transportation, hospitalization, surgical treatment, sudden dietary changes, administration of antimicrobials or anthelmintics, and other less well-defined eventsY Risk factors that can be extrapolated from other animal models or that have been strongly associated with human clostridial enterocolitis include advancing age, noninfectious agents such as nonsteroidal anti-inflammatory drugs, severity of underlying disease, period of hospitalization, intercurrent immunosuppressive disorder, use of rectal thermometers, enteral feeding, intestinal stasis, and gastrointestinal manipulation, including surgery.27 Typically, clostridial enterocolitis cannot be reproduced simply by administration of organisms or toxin(s).4H..02 Special conditions were required to produce enteritis experimentally in young foals by administering toxins or toxigenic clostridial strains by nasogastric tube. 28 In nearly all cases, some degree of
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disruption of the indigenous microflora is required as a predisposing event to overgrowth by toxigenic clostridia. Microbial Ecology of the Intestinal Tract
The barrier effect of the stable indigenous intestinal microflora that prevents the establishment and abundant growth of potentially pathogenic bacteria is referred to as "colonization resistance."·· 5.10,22,52 The bacterial flora of the ileum, cecum, and colon consist of hundreds of different species, of which more than 99% are anaerobes. Little is known about the complexity of interactions among the indigenous intestinal micro flora of the horse and specific consequences when they are disturbed. Disturbances of indigenous microflora may allow pathogenic or potentially pathogenic microorganisms to become established and multiply until they represent a larger proportion of the microbial population than would normally be the case. This occurs when pathogenic bacteria such as salmonella or spore-forming clostridia are resistant to an antimicrobial that has been administered to a patient. The theory of colonization resistance involves direct bacterial competition for adhesion to membrane-bound receptors, steric hindrance of other receptors when bound, production of antibacterial products, lowering of the pH through the production of volatile fatty acids (primarily acetic and lactic acids), and competition for substrates of metabolism. In addition to the microbial contributions to colonization resistance, host factors are also important. Normal peristalsis, epithelial turnover, and the mucus coating of the intestinal tract limit the adhesion of bacteria and toxins. '6 Colonization resistance is influenced by a range of factors, including management factors, general health status, intestinal motility and secretion, tissue oxygenation, exercise, and various forms of stress. Any factor that alters colonization resistance may shift the competitive growth balance in favor of toxigenic clostridia. Lysed bacteria (killed by antimicrobials) provide readily available nutrients for other bacteria. Decreased growth of certain bacteria may leave areas of the intestinal mucosa exposed, providing receptor sites for toxin or bacterial attachment. As opportunities become available for clostridia to grow, bacteria may be attracted from the lumen to the intestinal wall, where they produce and release toxins locally. Intestinal mucus of different animals has been shown to serve as a chemoattractant for C. difficile. s Adhesion to host tissue is important for full expression of virulence. It can be mediated by the fimbriae and physicochemical properties of the bacteria. During rapid growth, pathogenic clostridia produce an abundant array of hydrolytic enzymes and toxins that have the potential to degrade host tissue, contributing to existing pathologic changes and further compromising intestinal integrity. Clostridia likely derive nutritional benefit from the damaged tissue and subsequent fluid accumulation. s To gain further advantage, C. difficile is known to produce inhibitory metabolic products, including p-cresol, ammonia, and volatile fatty acids such as isocaproic acid.· Metabolic byproducts from other clostridia are also likely to enhance their own competitiveness in this milieu. The resulting alteration in microflora may also result in an increase in coliform count and more endotoxin production with resultant weakening of the mucosal barrier against endotoxin absorption as well as the direct tissue damage caused by clostridial factors. Antibiotic treatment is emerging as a factor commonly associated with acute enterocolitis. In some cases, it has been considered solely responsible for inducing acute colitis. 24 The risk of inducing enterocolitis is greatly increased by the use of certain antibiotics, especially when administered orally, in combination
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with the predisposing factors previously described and the presence of toxigenic Clostridium spp. Antibiotic-associated enterocolitis generally occurs within the first week after treatment is initiated, with the onset ranging from 1 to 8 days 2, 12, 24 Antimicrobials incriminated in enterocolitis include the f3-lactams (ampicillin, penicillin, and ceftiofur), erythromycin, metronidazole, aminoglycosides, and trimethoprim-sulfamethoxazole,lhlK The accidental ingestion of small amounts of erythromycin by mares during the treatment of their foals with this antibiotic was considered to be responsible for the changes in their intestinal microflora resulting in the development of acute colitis with peracute or acute onset and a fatal outcome in nine cases,'- 24 Interestingly, this problem has only been described in Sweden, It was considered to be such a serious problem that veterinarians replaced erythromycin with gentamicin for the treatment of R, equi infection in foals, without any further occurrence of the problem,3 The antimicrobials with the greatest risk of being associated with enterocolitis are those whose spectrum of activity includes anaerobes, Obligate anaerobic bacteria, which are considered to be the most important group responsible for maintaining colonization resistance, are usually susceptible to lincosamides, macrolides, f3-lactams, and tetracyclines, Treatment with these antibiotics has also been associated with enterocolitis in horses, which ranged from diarrhea to acute fatal colitis,2, n 3" The anaerobes are less susceptible to potentiated sulfonamides, fluoroquinolones, and aminoglycosides; as a result, the use of these antimicrobials is less frequently associated with diarrhea, Orally administered antimicrobials and those that undergo enterohepatic circulation or excretion into the intestine (tetracyclines, Iincosamid es, macrolides, and some cephalosporins) pose a greater risk of disrupting intestinal microflora than parenterally administered antimicrobials that do not gain access to the lumen of the intestine in an active form, DIAGNOSIS
The diagnosis of clostridial enterocolitis is based not on the clinical features of the disease but rathe r on the outcome of particular microbial and microbiologic laboratory examinations, The laboratory identification of Clostridium spp or toxins in feces from horses is usually considered to have diagnostic value, but achieving a definitive diagnosis is challenging and frequently impossible,12 Because various pathogenic clostridia and toxin types have been demonstrated in animals without signs of clostridial disease, such findings per se are not necessarily significant. The disruption of colonization resistance may allow the overgrowth of a toxigenic ClostridiulI1 sp without it necessarily contributing to the pathogenesis of disease, In most cases, microbiologic findings should be considered as providing supportive or presumptive evidence of a diagnosis. Confirmation of a diagnosis is most readily achieved when positive microbiologic findings can be correlated with lesions in the intestine that are consistent with the expected pathologic responses caused by the identified toxin or clostridial toxin type. For this reason, a definitive diagnosis of clostridial enterocolitis is frequently made only on postmortem examination ," Clostridium perfringens Diagnosis
The conventional diagnosis of C perfrillgclIs infection is based on isolation of the organism and demonstration of toxins in feces or intestinal contents, 12
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Some commensal carriage of C. pClfril1gells can be expected, howevel~ such that detection of organisms without evaluating their numbers or toxin production is of little diagnostic or prognostic value.,e " Most clinical laboratories expect to isolate C. perfrillgclIs using routine anaerobic media. Considerable variability exists among the culture methods used. On comparison of fiv e methods for culturing feces for C. pcrfril1gclls, the most sensitive method only detected 74% of the positive samples. In all cases, C. pClji-ingclls was d etected in at least one sample from which it was not isolated by any other method. It is thus necessary to use several methods when atte mpting cultivation of C. perti'illge1ls if the epidemiologic association between C. jiC/ji"ingeI15 and enterocolitis is to be accurately assessed and the predictive value of cultures for clinical diagnosis is to be validated." n Toxin detection and id entification are recommended diagnostic procedures to be used in conjunction with attempted isolation of C. jiclfrillgcl1s.' 7 Confirmatory evidence that toxins were produced by C. jicrfrillge1l s in vivo is based on identifying them in feces or intestinal contents . Toxin ne utrali za tion tests for detecting the major lethal toxins are not readily available, however. A few immunoassays for toxin detection are available primarily for detecting enterotoxin. ' The reverse passive latex (lgglutination assay (Oxoid PET-RPLA kit; Oxoid Inc., Ogdensberg, NY) is prone to giving a high rate of false-positive results. It is of little diagnostic value, because less than lO'Yo of the isolates from horses that tested positive by reverse passive latex agglutination assay were positive for the enterotoxin gene by PCR assay. II. " PCR multiplex assays for determining the toxin genotype of C. pClfrillgclls in isolates or fecal samples are currently the preferred diagnostic approach. ' ''·"" These ultrasensitive assays are unlikely to provide false-negative results unless some unrecognized toxin is present such as the recent discovery of the [32 toxin. It should be emphasized that positive findings do not diffe rentiate between the mere presence of the toxin genotype and the expression of toxin at biologically significant levels in the intestine. Results must be evaluated in light of other diagnosti c findings. Clostridium difficile Diagnosis
Diagnosis of C difficile infection requires isolation of the bacterium from feces or demonstration of cytotoxin in feces.' C. difficilc shedding by asymptomatic horses and foals at leve ls that are readily d etected by culture procedures is apparently rare. Because nontoxige nic strains are occasionally identified, toxin detection or testing of isola tes for toxigenicity is recommend ed. C. difficile is difficult to isolate, because culture techniques that e nhance germination of spores but prevent overgrowth by other enteric bacteria must be used. Media containing ingredients that enhance isolation and recognition of C. difficilc in hum an fecal samples are commercially available"' ; however, the optimum formulation of media for isolation of C. difficile from horses has not been critically evaluated at this time. In some cases, horses with acute colitis have been found to be culturepositive but not cytotoxin-positive until 1 or 2 days later2lS. " Because the detection of C. difficile infection in
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detection based on PCR amplification of the genes for toxins can be applied directly to fecal samples or isolated bacteria." 7, 22 These methods provide an increased sensitivity of between 10- and 100-fold over tissue culture detection of toxin. The direct PCR method may be most useful in elucidating the epidemiology of C. difficiZe infection by allowing detection of extremely low numbers of bacteria in specimens and the environment. Epidemiologic tracking of sources of infection can be accomplished by molecular analytic methods using a wide variety of markers that have identified over 400 unique types. 27 In addition, a variety of strains can be differentiated by antibiograms 22 and by serotyping based on cell wall antigens?
Sample Collection and Handling
As is the case with other bacterial infections, proper selection, collection, and transportation of clinical specimens are extremely important in optimizing the chances of laboratory diagnosis of clostridial enterocolitis.] For optimal results, a freshly collected fecal sample (ideally 20-30 mL) that has not become contaminated with environmental dust or soil is the preferred sample for culture or toxin assay. Swab specimens are inadequate for toxin assay, because the volume of fecal material obtained is too small. Swabs may be acceptable for epidemiologic investigations using sensitive assays (e.g., gene probes) or enrichment culture, where the objective is to determine carrier prevalence rather than to provide quantitative results that may aid in the interpretation of clinical significance and enable confirmation of a diagnosis. To optimize bacterial isolation or detection of toxins, samples should be processed within 2 hours of collection. Sporulation rates, survival of vegetative clostridial cells, and stability of fecal toxins vary greatly among the clostridia; thus, recommendations for acceptable handling of a specimen for one assay are not necessarily optimal for all possible assays. Although spores survive for several days in refrigerated samples, a large decrease in the number of viable vegetative cells of clostridia can be expected. Prolonged storage of fecal samples at ambient or refrigerated temperatures is also likely to lead to denaturation of clostridial toxins. Freezing samples at - 70°C is thus routinely recommended for optimal long-term preservation of samples for culture and toxin detection. There is a critical need for comprehensive evaluation of the diagnostic methods used for detection of clostridial enterocolitis in horses that would establish the optimum number and periodicity of fecal sampling for clostridial culture and toxin detection.]7 Although little clinically useful information is gained by repeating assays for C. difficile within 7 days in humans/, the same recommendation does not seem to apply to horses. Multiple samples must be collected, because the nonhomogeneous nature and volume of equine intestinal contents and feces may not permit ready detection of the organism. Early in the course of infection, clostridia may be more closely associated with mucosal surfaces and may not be found in feces. Feces can continue to be normal in appearance even though the animal may be exhibiting peracute signs of enterocolitis. The diagnostic challenge of detecting and associating C. difficile with equine clostridial enterocolitis is well illustrated by reports where the organism or its toxins were not detected until late in the course of the disease after up to 5 days of daily serial sampling. 45, ,. Daily fecal samples should thus be collected and submitted to the laboratory until the clinical signs of disease begin resolving or a positive result is obtained.
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TREATMENT
The fatality rate for clostridial enterocolitis is highly variable (10%-70%) among treated horses and depends on the virulence of the pathogen, susceptibility of the host, and aggressiveness of the therapy." Horses with acute enterocolitis may require a combination of fluid therapy, nonsteroidal anti-inflammatory drugs, antidiarrheal agents, antimicrobial agents, dietary management, and other supportive care as previously reported in the literature. 13 This discussion is limited to treatment options for the clostridial component of the acute enterocolitis syndrome. Antimicrobial Treatment
Antimicrobial drugs with activity against clostridial organisms that are appropriate for use in the horse include bacitracin, metronidazole, and vancomycin. Oral bacitracin has been recommended based on the treatment and prevention of idiopathic colitis induced in horses, which is presumably caused by unidentified Clostridium Spp.S1l In vitro determination of the antimicrobial susceptibilities of 105 C. difftcile isolates from horses revealed that all were resistant to greater than 1024 fLg of bacitracin per milliliter.co It seems that bacitracin is not a logical first-line drug of choice for treating clostridial enterocolitis if C. difficile is possibly involved. 54 Successful treatment of equine clostridial enterocolitis with metronidazole has been reported. Statistically, metronidazole improved the survival rate for acute nosocomial enterocolitis presumed to be caused by clostridia. 3" A 10-day course of metronidazole treatment was associated with clinical improvement and full recovery in a mare with acute enterocolitis attributed to infection with C. difficile and C. perfringens. 1h Up to 40% of C. difftcile isolates from horses at one veterinary hospital were classified as resistant to metronidazole, however, based on in vitro antimicrobial susceptibility testing. 2b , 35 Insufficient data are available from clinical trials in horses to determine the efficacy or complications of metronidazole treatment or to provide a definitive antimicrobial treatment recommendation for clostridial enterocolitis, There are no statistically significant differences in the response or relapse rates for oral vancomycin compared with oral metronidazole treatment in human patients. 55 Nevertheless, metronidazole is considered to be the first-line drug of choice, because vancomycin is much more expensive and poses a greater risk of selecting glycopeptide resistance in other pathogens such as enterococciY Despite appropriate antimicrobial therapy, approximately 15°/r, to 25% of human patients experience relapses of C. difficile enterocolitis once treatment is discontinued. At least half of these putative relapses are, in fact, cases of reinfection with a different strain of C. difficile. 55 Because the vegetative cells of clostridia are susceptible to antimicrobial drugs, the presence of spores may be responsible for an apparent lack of response to treatment. This emphasizes the fact that susceptibility to new clostridial strains with reinfection due to impaired colonization resistance continues until the underlying predisposing deficits are corrected. PREVENTION Immunization
Vaccines with proven safety and efficacy to reduce the risk of clostridial enterocolitis have not been developed for use in horses. C. perfringens type C
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and D toxoid developed for ruminants has been administered to horses, but safety and efficacy data are not available.'" '2 There are no vaccines against C. difficile. The frequency of relapses and chronic C. difficilc infections in humans suggests that the protective value of convalescent immunity may be limited. Lack of animal models and the inability to consistently reproduce disease by oral administration of organisms or toxins are major factors limiting efficacy testing of proposed vaccines. One promising approach to immunotherapy has been the production of an anti-C. difficile bovine immunoglobulin concentrate for oral administration, which is reported to inhibit cytotoxicity and enterotoxicity.55
Biotherapy
Biotherapy aims to restore the commensal intestinal micro flora and thus optimize colonization resistance against Clostridium spp. In 15'Yo to 25% of human patients, cessation of antimicrobial therapy is all that was required to re-establish colonization resistance." Although a variety of probiotics (bacterial and yeast products) have been administered to humans and evaluated in various laboratory animal models and in vitro systems, efficacy data in horses are lacking. Commercial preparations of Saccharomyces boulardii have been shown to have significant therapeutic and prophylactic potential in humans for reducing diarrhea and recurrences associated with C. difficile infection.OJ 00 S. lJOulardii was shown to prevent binding of C. difficile enterotoxin in a rat model. It should be noted that S. cerevisiae (brewer's yeast) is distinct from S. boulardii and should not be considered equivalent to a commercial preparation of S. boulardii. A combination of Lactobacillus and xylitol has also been shown to have a protective effect, possibly by inhibiting the adhesion and growth of C. difficilc. 4 ()
SUMMARY
Equine clostridial enterocolitis is being recognized with increasing frequency. It has been identified in foals with diarrhea, antibiotic-associated enterocolitis, or nosocomial enterocolitis. The sporadic occurrence of clostridial enterocolitis, the variety of types of clostridia involved, and the difficulty of experimentally reproducing the disease suggest that it is a poorly defined multifactorial syndrome. The risk factors associated with susceptibility to colonization and progressive infection are largely based on anecdotal observations and extrapolation from human studies. Quantitative studies are needed to decipher the complex interactions between host and indigenous microflora that provide for and maintain a healthy colonization resistance environment. It seems that such studies might be more beneficial in furthering our understanding of the pathogenesis of clostridial enterocolitis than attempting to implicate another agent or toxin as the sole cause of the disease in equids. Treatment protocols that interrupt the pathogenesis of the disease need to be devised and critically evaluated to complement the present protocols emphasizing supportive care. Perhaps it is time to consider clostridial enterocolitis as yet another consequence of the use of antimicrobials analogous to the selective pressures that result in the emergence of multiple drug-resistant pathogens.
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References 1. Allen SO, Emery CL, Siders JA: Clostridiu11l. 111 Murray PR (ed): Manual of Clinical Microbiology, ed 7. Washington, DC American Society for Microbiology, 1999, pp 654~ 671 2. Baverud V, Gustafsson A, Franklin A, et al: Clostridiu1l1 difficile associated with acute colitis in mature horses treated with antibiotics. Equine Vet J 29:279~284, 1997 3. Baverud V, Franklin A, Gunnarsson A et al: ClostridiulII difficile associated with acute colitis in mares when their foals are treated with erythromycin and rifampicin for Rhodococcus cqui pneumonia. Equine Vet J 30:482--488, 1998 4. Bongaerts GP, Lyerly OM: Role of bacterial metabolism and phYSiology in the pathogenesis of Clostridhllli dijJicile disease. Microb Pathog 22:253~256, 1997 5. Borriello SP: Pathogenesis of Clostridium dijJicile infection. J Antimicrob Chemother 41(suppl C):13~19, 1998 6. Brazier JS: The diagnosis of Clostridiulll dijJicile~associated disease. J Antimicrob Chemother 41(suppl C):29~40, 1998 7. Brazier JS: The epidemiology and typing of ClostridiulIl difficile. J Antimicrob Chemother 41(suppl C):47~57, 1998 8. Browning GF, Chalmers RM, Snodgrass DR, et al: The prevalence of enteric pathogens in diarrhoeic Thoroughbred foals in Britain and Ireland. Equine Vet J 23:405-409, 1991 9. Bueschel 0, Walker R Woods L, et al: Enterotoxigenic ClostridiulII perfringens type A necrotic enteritis in a foal. JAVMA 213:1305~1307, 1998 10. Butel MJ, Roland N, Hibert A, et al: Clostridial pathogenicity in experimental necrotising enterocolitis in gnotobiotic quails and protective role of bifidobacteria. J Med Microbiol 47:391~399, 1998 11. Chanter N, Netherwood T, Wood JLN, et al: A case-control study of foal diarrhoea, in particular investigating the role of Clostridiull1 perfril1gells. Epidemiol Sante Anim 3132:03.05.1~03.05.3, 1997 12. Cohen NO, Divers TJ: Acute colitis in horses. Part 1. Assessment. Com pend Contin Educ Pract Vet 20:92~98, 1998 13. Cohen NO, Divers TJ: Acute colitis in horses. Part II . Initial management. Compend Contin Educ Pract Vet 20:228~234, 1998 14. Cosmetatos" Perrin L Gallusser A, et al: Faecal isolation of Clostridiulll difficile and its toxins from horses with typhlo-colitis. III Equine Infectious Diseases Vll: Proceedings of the Seventh International Conference, Tokyo, 1994, P 363 15. Daube G, Simon P, Limbourg B, et al: Hybridization of 2,659 Clostridium perfringens isolates with gene probes for seven toxins (a, ~, E, L, 6, fl, and enterotoxin) and for sialidase. Am J Vet Res 57:496~501, 1996 16. Davis MS, Murray MJ, Carman RJ: Diagnosis and treatment of clostridial colitis in a mare. Vet Med (Praha) 94:363~366, 1999 17. Donaldson MT, Palmer JE: Prevalence of Clostridiul1l perfril1gcl1s enterotoxin and Clostridium difficile toxin A in feces of horses with diarrhea and colic. JAVMA 215:358~ 361, 1999 18. East LM, Savage C), Traub-Dargatz JL, et al: Enterocolitis associated with Clostridium perfrillgells infection in neonatal foals: 54 cases (1988~1997). JAVMA 212:1751~1756, 1998 19. Gibert M, Jolivet-Renaud C Popoff MR: Beta2 toxin, a novel toxin produced by Clostridiulll perfringens. Gene 203:65~73, 1997 20. Ginter A, Williamson ED, Dessy F, et al: Molecular variation between the a-toxins from the type strain (NCTC 8237) and clinical isolates of Clostridiulll pelfrillgens associated with disease in man and animals. Microbiology 142:191~198, 1996 21. Griffiths NJ, Walton JR, Edwards GB, et al: The prevalence of ClostridiUlll pClfrillgelis in the horse. Reviews in Medical Microbiology 8(suppl):S52~54, 1997 22. Groschel OHM: Clostridium difficile infection. Crit Rev Clin Lab Sci 33:203~245, 1996 23. Guerrant RL, Steiner TS, Lima AAM, et al: How intestinal bacteria cause disease. J Infect Dis 179(suppl):S331 ~337, 1999 24. Gustafsson A, Baverud V, Gunnarsson A, et al: The association of erythromycin ethylsuccinate with acute colitis in horses in Sweden. Equine Vet J 29:314~318, 1997
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25. Herholz e, Miserez R, Nicolet J, et al: Prevalence of 132-toxigenic Clostridium pcrfrillgens in horses with intestinal disorders. J Clin Microbiol 37:358-361, 1999 26. Jang SS, Hansen LM, Breher JE, et al: Antimicrobial susceptibilities of equine isolates of Clostridium difficile and molecular characterization of metronidazole-resistant strains. Clin Infect Dis 25(suppl):S266-267, 1997 27. Johnson S, Gerding DN: Clostridium difficile-associated diarrhea. Clin Infect Dis 26:1027-1034, 1998 28. Jones RL, Shideler RK, Cockerell GL: Association of ClostridiulIl difficile with foal diarrhea. III Equine Infectious Diseases V: Proceedings of the Fifth International Conference, Lexington, 1989, pp 236-240 29. Jones RL, Adney WS, Shideler RK: Isolation of Clostridium difficile and detection of cytotoxin in the feces of diarrheic foals in the absence of anti-microbial treatment. J Clin Microbiol 25: 1225-1227, 1987 30. Jones RL, Adney WS, Alexander AF, et al: HemorrhagiC necrotizing enterocolitis associated with ClostridiuJll difficile infection in four foals. JAVMA 193:76-79, 1988 31. Jones SL, Wilson WD: ClostridiulII septicuIII septicemia in a neonatal foal with hemorrhagic enteritis. Cornell Vet 83:143-151,1993 32. Kanoe M, Inoue S, Abe T, et al: Isolation of ClostridiuJll perjril/gens from foals. Microbios 64:153-158,1990 33. Larsen J: Acute colitis in adult horses: A review with emphasis on aetiology and pathogenesis. Vet Q 19:72-80, 1997 34. Madewell BR, Tang YJ, Jang S, et al: Apparent outbreaks of ClostridiulII difficileassociated diarrhea in horses in a veterinary medical teaching hospital. J Vet Diagn Invest 7:343-346, 1995 35. Madigan JE, Magdesian G, Barlough J, et al: Recent developments on infectious colitis in horses: Clostridiul11 difficile and Elzrlichia risticii (Potomac Horse Fever). Schweiz Arch Tierheilkd 140:469-470, 1998 36. Magdesian KG, Madigan JE, Hirsh De, et al: Clostridium dijficile and horses: A review. Rev Med Microbiol 8(suppl):S46-48, 1997 37. McClane BA: New insights into the genetics and regulation of expression of Clostridium perjringens enterotoxin. Curr Top Microbiol Immunol 225:37-55, 1998 38. McGorum Be, Dixon PM, Smith DGE: Use of metronidazole in equine acute idiopathic toxaemic colitis. Vet Rec 142:635-638, 1998 39. Meer R, Songer JG: Multiplex polymerase chain reaction assay for genotyping Clostridium perfringens. Am J Vet Res 58:702-705, 1997 40. Naaber P, Mikelsaar RH, Salminen S, et al: Bacterial translocation, intestinal microflora and morphological changes of intestinal mucosa in experimental models of ClostridiulIl difficilf infection. J Med Microbiol 47:591-598, 1998 41. Netherwood T, Chanter N, Mumford JA: Improved isolation of ClostridiulIl perfrillgens from foal faeces. Res Vet Sci 61:147-151, 1996 42. Netherwood T, Binns M, Townsend H, et al: The ClostridiulII perjringens enterotoxin from equine isolates: Its characterization, sequence and role in foal diarrhoea. Epidemiol Infect 120:193-200, 1998 43. Netherwood T, Wood JLN, Townsend HGG, et al: Foal diarrhoea between 1991 and 1994 in the United Kingdom associated with Clostridium perjringens, rotavirus, StrOlzgyloides westeri and Cryptosl'oridilllll spp. Epidemiol Infect 117:375-383, 1996 44. Netherwood T, Wood JLN, Mumford JA, et al: Molecular analysis of the virulence determinants of Clostridilllll pClfringcns associated with foal diarrhoea. Vet J 155:289294, 1998 45. Perrin J, Cosmetatos I, Gallusser A, et al: Clostridillm difficilc associated with typhlocolitis in an adult horse. J Vet Diagn Invest 5:99-101, 1993 46. Sage R: Nosocomial infections: Listening to human experience may help the horse. Equine Vet J 30:450-451, 1998 47. Saif NA, Brazier JS: The distribution of Clostridiulll ditficile in the environment of South Wales. J Med Microbiol 45:133-137, 1996 .. 48. Songer JG: Clostridial enteric diseases of domestic animals. C1in Microbiol Rev 9:216234, 1996
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49. Staempfli HR, Prescott JF, Brash ML: Lincomycin-induced severe colitis in ponies: Association with Clostridium clldl/veris. Ca n J Vet Res 56:168- 169, 1992 50. Staempfli HR, Prescott JF, Carman RJ, et al: Use of bacitracin in the prevention and treatment of experimentally-induced idiopathic colitis in horses. Can J Vet Res 56:233- 236, 1992 51. Teale C), Naylor RD: ClostridiulIl dijficile infection in a horse. Vet Rec 142:47, 1998 52. Traub-Dargatz JL, Jon es RL: Clostridia associated enterocolitis in adult horses and foals. Vet Clin North Am Equine Pract 9:411-421,1993 53. Tschirdewahn B, Notermans S, Werna rs K, et al: The presence of enterotoxigenic Clostridiu111 perfrillgclls strains in faeces of various animals. lnt J Food Microbiol 14:175178,1992 54. Weese JS, Parsons A, Staempfli HR: Association of ClostridiulIl difficile with enterocolitis and lactose intolerance in a foal. JAVMA 214:229-232, 1999 55. Wilcox MH: Treatment of Clostridiu111 difficile infection. J Antimicrob Chemother 41(suppl C):41-46, 1998
Address repril1t requests to Robert L. Jones, DVM, PhD Department of Microbiology Colorado State University Fort Collins, CO 80523 e-mail: [email protected]
0749- ()739 / lIO $15.00 + .00
CLOSTRIDIAL ENTEROCOLITIS Robert L. Jones, DVM, PhD
Acute enterocolitis and diarrhea associated with clostridia are garnering greater recognition as important clinical problems. An e lectronic search for references on equine clostridial entemcolitis illustrates the increasing significance of the disease in the 19905. This is evidenced by 9 citations in the 1970s, 19 in the 1980s, and nearly 50 in the 1990s that deal with epidemiology, virulence factors, improved diagnostic tests, and evaluation of treatment outcomes. Based on these studies, clostridia have been firmly incriminated as playing a major causative role in enterocolitis in horses. Clostridium pClji'illgCIIS is associated with diarrhea in foals and enterocolitis in horses of all ages. C. difficile has eme rged as an important cause of nosocomial and antibiotic-associated enterocolitis in horses. Included among the reasons that may account for the emergent role of clostridia in enterocolitis and diarrhe,l are an increased awareness among owners and veterinarians resulting in more detailed and comprehensive investigations, increased sensitivity of diagnostic assays, changing management conditions that modify natural resistance to this syndrome, and the impact of antimicrobial use and resistance on the initiation of novel clinical problems. The genus ClostridiunI includes many species of obligatory anaerobic to aerotolerant spore-forming gram-positive rods. Because sporulation increases resistance to drying, heat, irradiation, and chemicals, clostridia are generally ubiquitous in nature, existing principally in soil and the intestinal tracts of many animals. C. peljrillgclI s and C. difficile are the species most frequently isolated from cases of equine enterocolitis. C. scptiCllIII," C. sordelli," C. Cildaveris,'9. '" C. paraputrificulII,5I1 and at least six unidentified clostridia'" have been recovered from acute enterocolitis in the horse. In addition, C. spirojormc,"' C. botulinulIl types C and D producing C2 toxin,''' and C. butyricwn'" have bee n associated with acute enterocolitis in other species. Although exogenous spread from animal to animal has been confirmed during outbreaks of disease, endogenous infections involving clostridia that a re a part of the host's own microflora are probably much more common. The basic biology of clostridia and the clinical
From the Depa rtment of Microbiology, College of Vete rinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado
VETERINARY CLINICS OF NORTH AMERICA: EQUINE PRACTICE VOLUME 16· NUMBER 3 · DECEMBER 21100
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management of enterocolitis have been described in detail.lJ, 33,10, 4", 52 In this article, emphasis is placed on recent advances in our understanding of the disease, including the discovery of C. perfringens (32 toxin, the epidemiology of C. perfringens, nosocomial and antibiotic-related enterocolitis associated with C. difficile, and the effect of factors such as antibiotic use and hospitalization on the occurrence of equine enterocolitis.
CLOSTRIDIUM PERFRINGENS C. perfringens, also called C. welchii, is phenotypically and genetically highly variable; this provides for differences in virulence among strains and in their ability to cause disease. C. perfringens is a prolific toxin producer that is capable of expressing as many as 17 exotoxins, although individual strains produce only a defined subset of these toxins. 1, 37, 4",52 This characteristic forms the basis for a toxin-typing system for classifying C. perfringells isolates into five types (A, B, e, D, and E) based on an isolate's ability to produce one or more of the "major lethal" toxins. Other varieties or subtypes have been described within or in addition to these types based on the production of apparently less important toxins. Other typing systems are based on a strain's ability to produce enterotoxin, antigenic relatedness based on cell wall antigens (serotypes), and molecular types or genotypes based on nucleic acid analysis.
Prevalence of Clostridium perfringens C. perfringens is widely distributed as vegetative cells and spores in soil and fecal matter, which ensures that the organism is frequently present on surfaces exposed to dust contamination, including many feed items.' C. perfringens is one of the first organisms to colonize the neonatal intestinal tract, and a proportion of any healthy population of mammals can be expected to carry the organism." Most reports probably underestimate the true prevalence of C. perfringens in the intestinal tract of horses and do not differentiate between organisms in transit through the tract and those that are permanently established as part of the indigenous microflora. Geographic and seasonal variations in environmental concentrations of C. perfringens undoubtedly alter the quantity and shedding of this bacterium. Furthermore, differences in sampling and detection methods may explain the disparity in prevalence that has been reported in different studies. C. perfringens may exist in one or more states; these include the vegetative cell, the sporulating cell, the endospore, and the germinating endospore, each of which requires unique culture conditions to optimize the chances of recovery.41 When detected in the feces of healthy horses, C. perfrillgens is usually isolated only in small numbers. A count of less than 103 colony forming units (cfu) per gram of feces is considered normal,n, 33 with 75% of positive fecal cultures yielding less than 100 cfu / g." Using relatively insensitive direct plating methods, C. perfrillgens was detected in feces from 2 of 32 (6'Yo) healthy foals" and 12 of 50 (24%) horses. 53 When using a combination of direct plating, enrichment, and spore selection methods, C. perfringens was isolated from the feces of 4 of 29 (14%) healthy foals, 32 23 of 124 (19%) foals," 60 of 223 (27%) foals,.3 173 of 271 (64%) foals,"1 and 77 of 105 (73%) healthy horses."
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Association of Clostridium perfringens with Enterocolitis
The presence of C. perfringens in large numbers in feces and intestinal contents has been associated with acute equine enterocolitis in Europe 33 and North America.'"' 52 C. perfringells was isolated from cases of diarrhea from 12 of 304 (4%) foals/ and 58 of 365 (16%) foals," and from 57% of 421 foals and 27% of 223 healthy foals. 41 It was associated with 68'1'0 of 22 foals with diarrhea that died,4344 and'with 47 of 54 foals with rapidly progressive enterocolitis that often had a fatal outcome, despite intensive supportive therapy." C. perfringens was isolated from 95°;\, of samples obtained from horses with anterior enteritis compared with 60')(, of samples from other types of COlic. 21 Subtyping the 165-235 intergenic spacer revealed a new genotype of C. perfringells that seems to playa major pathogenic role in foal diarrhea. It was isolated from 12 of 14 fatal cases, but the toxin type was not identified." Toxin Types of Clostridium perfringens
The major toxin produced by C. perfringells type A is a toxin; this is also produced in varying amounts by all typeable strains but not consistently in lethal quantities by some type A strains. The a toxin has both phospholipase C and sphingomyelinase activities and is able to elicit a variety of subtle effects on eukaryotic cell metabolism, including activation of the arachidonic acid cascade and stimulation of protein kinase C activity.211 There is direct evidence for a toxin activity in toxic infections of tissue 211 Although type A is the predominant C. perfrillgells type isolated from healthy horses, II, 21 foals with diarrhea, 11,12,41 or foals and adult horses with enterocolitis,'J.2! there is little direct evidence incriminating this toxin as a significant enteric virulence factor." A variant of a toxin that is more resistant to intestinal chymotrypsin than the toxin from nongastrointestinal disease isolates has been identified/" suggesting that it may have some role in enterocolitis. The presence of hemorrhagic necrotizing lesions in some foals 9 from which type A is isolated suggests that unidentified factors other than a toxin or enterotoxin are contributing to the pathogenesis of the intestinal lesions. A novel toxin produced by C. perfrillgells and recently designated as (32 toxin may play an important role in the pathogenesis of necrotizing intestinal disorders in horses.!9 25 Both (32 and (3 toxins have comparable biologic activities, with each capable of causing hemorrhage and necrosis of the intestinal wall, lethal infection in mice, and cytotoxicity in selected cell cultures. The (32 and 13 toxins lack significant amino acid identity; this results in limited antigenic crossreactivity. The (32 toxin gene was found in 15 isolates of C. perfrillgells recovered in high numbers (>10" cfu I g) from the intestinal contents of horses that died with enterocolitis. I'> In a second survey,25 an undesignated toxin type of C. perfringens containing the a and 132 toxin genes was found in 75'X, of samples of intestinal contents and biopsy specimens of the intestinal wall from horses with acute enterocolitis. It was found in a lower percentage (62%) of horses with other intestinal disorders but was not recovered at all from the feces of 58 control horses. Although currently unproven, the newly identified (32 toxin, which was present in isolates from horses that would otherwise have been classified as type A, may play an important role as a cytotoxin in the pathogenesis of toxic intestinal disorders in horses. Some C. perfi'illgells strains produce enterotoxin, a virulence factor best known as a cause of food poisoning in human beings. Enterotoxin causes
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cell membrane pore formation, followed by altered permeability, inhibition of macromolecular synthesis, cytoskeletal disintegration, and lysis of cells. Only 2% to 6% of the global C. perfringells population carries the enterotoxin gene, and these isolates are almost always type A.37 Enterotoxigenic strains of C. perfringens were found in feces of 7 of 50 (14'Yo) horses," and in 13% of horses l5 in general surveys. In one study, enterotoxigenic strains were found in 16'X) of horses with diarrhea but not in age- and seasonally matched controls. I? In cases of foal diarrhea, enterotoxigenicity was no more common among isolates than in matched controls. The prevalence of enterotoxin-positive strains of C. perfringens as determined by polymerase chain reaction (PCR) assay was too low (9.7%) for them to be considered a pathogenic subtype. II . '2 Several authors have discounted the importance of enterotoxin in the pathogenesis of C. pelfrillgensassociated foal diarrhea, II. 17.33, Sil although others consider that it may still have a minor role to play in cases of foal diarrhea that resolve but not in those that have a fatal outcome. 42 C. perfringens types B, C, and D have been associated with severe necrotizing, hemorrhagic enterocolitis in foals. 4K , ,2 Type C is the most commonly reported clostridial enteric pathogen in foals in North America. IK,", The [) toxin of types Band C induces inflammation and progressive necrosis of intestinal mucosa with epithelial cell death and desquamation, followed by further bacterial invasion, multiplication, and even more toxin production!' Lesions found in the jejunum and ileum usually consist of acute hemorrhagic enteritis with necrosis of villi, with large numbers of gram-positive rods demonstrable in stained smears and sections. If diarrhea is present, it usually occurs for only a brief period before death. The precise biologic activity of the E toxin of C. perfringens type D, which increases intestinal permeability and causes toxicity of the central nervous system, has not been identified. There is limited evidence that this toxin type can be a significant pathogen in horses.
CLOSTRIDIUM DIFFICILE C. difficile is currently the most frequently identified cause of nosocomial and antibiotic-associated diarrhea in human beings, even though it can only be isolated and identified in approximately 25% of cases! The pathogenesis of C. difficile infection in humans has been the subject of several extensive reviews. I, " 5,17,22,27,36,4" C. difficile produces several hydrolytic enzymes and at least five toxic factors.' Only toxin A, which is primarily an enterotoxin, and toxin B, a cytotoxin, have been studied in sufficient detail to confirm their involvement in enterocolitis. s Ingested spores survive the low pH of the stomach and upper small intestine, germinate in the terminal ileum, and multiply in the colonic lumen. In the absence of competition by the indigenous microflora (colonization resistance), C. difficile may grow to high numbers (>10" cfu / g). The critical time in terms of susceptibility to infection is the period just after reduction of the colonic flora and before they are re-established." As C. difficile increases in number, it produces toxins and enzymes that damage tissue. When these toxins are produced, they act synergistically to induce damage to intestinal tissue and disrupt cell-to-cell tight junctions, resulting in acute inflammation, mucosal permeability, fluid accumulation, and necrosis of the epithelium. s 23 Released nutrients, and possibly locally induced anoxia, may, in turn, stimulate C. difficile growth and toxin production. Ultimately, this can lead to cell detachment, local necrosis, hemorrhage, and pseudomembranous lesions. In humans, the result of
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C. difficile infection can range from asymptomatic colonization to severe diarrhea, pseudomembranous colitis, toxic megacolon, colonic perforation, and death." 22, 27 Epidemiology of Clostridium difficile Infections
The heat- and acid-resistant spores of C. difficile are capable of persisting for months to years in the environment.' " C. difficile has been found inconsistently in the environment, suggesting a patchy environmental distribution or more probably a reflection of variable sampling methodologies and insensitive culture methods. 7 In South Wales, C. ditJicile was found in 7'1,) of samples from water (river, sea, and lake) and soil, 1'Yo of horse feces, and 17°;\, of environmental samples from veterinary clinics.· 7 In humans, the consensus is that 1% to 2'Yo (range, 0%-15%) of adults and up to 60% of healthy neonates carry C. difficile.'6 Animal reservoirs have been recognized, and a great variety of wild, farm, and domestic species may carry the organism. 7 It is not known if intestinal carriage is a temporary or permanent state in animals and humans. Carriage of C. difficile, even in large numbers, is not associated with increased risk of disease. Although person-to-person spread has been considered the likely source of infection for most documented outbreaks of C. difficile enteritis in hospitals, the environment and endogenous carriers are increasingly being suspected as sources of infection and reinfection'" for some cases of diarrheal disease in the general community." In 1987, toxigenic C. difficile was first implicated as a cause of equine enterocolitis in a group of foals with diarrhea'4 and in a series of foals with hemorrhagic necrotizing enteritis. 3 Within the past decade, numerous studies have provided evidence of the causative role of C. difficile in nosocomial diarrhea and acute colitis' 3,1-1, , . " 36, .h, 5 1. 3·) as well as in antibiotic-associated diarrhea and enterocolitis of adult horses,",3. 1.,2 1 .5 C. difficile is infrequently isolated from clinically normal adult horses and foals. There is some suggestion that it might be more prevalent in horses than isolation rates would indicate. 22 When identified, the prevalence is usually less than 2%2,3,24,,. ,6. 43 It is quite likely that the prevalence of C. difficile carriers would be much greater if more sensitive culture methods or molecular detection methods such as PCR assay were used, however. The prevalence and risk factors for C. difficile infection in foals have not been established. Some foals develop diarrhea without a history of antimicrobial treatment/" whereas others become asymptomatic carriers and potential reservoirs of C. difficilc after antimicrobial treatment.' Other factors that require evaluation in connection with C. difficilc infection include intercurrent rota virus infection"l and lactose intolerance. s• In 1993, C. ditJicile was first associated with enterocolitis in an adult horse.·' Since then, C. difficile has been associated with major outbreaks of nosocomial enterocolitis in horses in a number of equine hospitals in Europe 3, 1.,51 and the United States,'"' '6 leading one author to suggest that most hospitals experience sporadic episodes of the disease.·" Prior antimicrobial use (systemic, oral, or both) was a common factor in the history of most of these cases. 2• 14, 34-3<> The importance of antibiotic use as a risk factor was supported by findings from Sweden, where C. difficile or its cytotoxin was identified in fecal samples from 5 of 11 mares that developed acute colitis when their foals were treated orally with erythromycin and rifampicin for Rhodococcus equi infection. 3 C. difficile or its cytotoxin has been identified in the feces and intestinal contents of adult horses with acute colitis or diarrhea in several other studies. 16 - IH, 25, .X The nature of the interactions between C. difficilc and hospitalization, antimicrobial treat(1
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ment, and preexisting gastrointestinal disease have not been determined. C.
difficile has been associated with acute colitis and diarrhea in adult horses sufficiently frequently, however, for several authors to recommend routine testing for C. difficile and its cytotoxin in any horse that presents with acute colitis and diarrhea after treatment with antibiotics or in association with hospitalization.I. 3. 16
CLINICAL FEATURES
Clostridial enterocolitis occurs in horses worldwide, ranging in severity from asymptomatic to acute fulminating enterocolitis with a high case fatality rate. It usually occurs on a sporadic basis; however, occasionally, there can be clusters of cases in foals on breeding farms and nosocomial outbreaks. There seem to be two major manifestations of clostridial enterocolitis in foals: diarrhea with mild enteritis and severe hemorrhagic necrotizing enterocolitis. In respect to the former, diarrhea is the primary clinical sign, and whereas supportive treatment can be beneficial, many foals make spontaneous recoveries."· 43 Because of this, the incidence of this form of clostridial disease is probably underreported. The more severe form of clostridial enterocolitis presents as a rapidly progressive disease that often has a fatal outcome, despite the implementation of intensive care measures. Foals typically present with diarrhea (approximately 50% may have hemorrhagic diarrhea), obtunded mentation, clinical dehydration, colic, and tachypnea. '8 Acute enterocolitis in adult horses varies from moderate to severe, with diarrhea ranging from loose pasty to watery hemorrhagic feces, accompanied by abdominal pain, fever, d ecreased appetite, septic shock, and even sudden death.l2· 33 The onset of clinical signs is usually rapid but may extend over a period of several days in some cases. Evidence of dehydration and shock is evident on postmortem examination. The intestines may be distended with fluid, and discoloration of the serosa is frequently present. Necrosis of the mucosa may range from superficial to deep and may be accompanied by mild mucosal edema to a severe fibrinous hemorrhagic exudate on the mucosal surface. EPIDEMIOLOGY
Many horses are temporary or permanen t enteric carriers of clostridia. Some of these animals are susceptible to disease, although others seem to remain resistant to disease for extended periods, during which they remain culturepositive for these organisms. A number of risk factors are thought to be involved in equine clostridial enteritis. These include stressful events such as racing, transportation, hospitalization, surgical treatment, sudden dietary changes, administration of antimicrobials or anthelmintics, and other less well-defined eventsY Risk factors that can be extrapolated from other animal models or that have been strongly associated with human clostridial enterocolitis include advancing age, noninfectious agents such as nonsteroidal anti-inflammatory drugs, severity of underlying disease, period of hospitalization, intercurrent immunosuppressive disorder, use of rectal thermometers, enteral feeding, intestinal stasis, and gastrointestinal manipulation, including surgery.27 Typically, clostridial enterocolitis cannot be reproduced simply by administration of organisms or toxin(s).4H..02 Special conditions were required to produce enteritis experimentally in young foals by administering toxins or toxigenic clostridial strains by nasogastric tube. 28 In nearly all cases, some degree of
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disruption of the indigenous microflora is required as a predisposing event to overgrowth by toxigenic clostridia. Microbial Ecology of the Intestinal Tract
The barrier effect of the stable indigenous intestinal microflora that prevents the establishment and abundant growth of potentially pathogenic bacteria is referred to as "colonization resistance."·· 5.10,22,52 The bacterial flora of the ileum, cecum, and colon consist of hundreds of different species, of which more than 99% are anaerobes. Little is known about the complexity of interactions among the indigenous intestinal micro flora of the horse and specific consequences when they are disturbed. Disturbances of indigenous microflora may allow pathogenic or potentially pathogenic microorganisms to become established and multiply until they represent a larger proportion of the microbial population than would normally be the case. This occurs when pathogenic bacteria such as salmonella or spore-forming clostridia are resistant to an antimicrobial that has been administered to a patient. The theory of colonization resistance involves direct bacterial competition for adhesion to membrane-bound receptors, steric hindrance of other receptors when bound, production of antibacterial products, lowering of the pH through the production of volatile fatty acids (primarily acetic and lactic acids), and competition for substrates of metabolism. In addition to the microbial contributions to colonization resistance, host factors are also important. Normal peristalsis, epithelial turnover, and the mucus coating of the intestinal tract limit the adhesion of bacteria and toxins. '6 Colonization resistance is influenced by a range of factors, including management factors, general health status, intestinal motility and secretion, tissue oxygenation, exercise, and various forms of stress. Any factor that alters colonization resistance may shift the competitive growth balance in favor of toxigenic clostridia. Lysed bacteria (killed by antimicrobials) provide readily available nutrients for other bacteria. Decreased growth of certain bacteria may leave areas of the intestinal mucosa exposed, providing receptor sites for toxin or bacterial attachment. As opportunities become available for clostridia to grow, bacteria may be attracted from the lumen to the intestinal wall, where they produce and release toxins locally. Intestinal mucus of different animals has been shown to serve as a chemoattractant for C. difficile. s Adhesion to host tissue is important for full expression of virulence. It can be mediated by the fimbriae and physicochemical properties of the bacteria. During rapid growth, pathogenic clostridia produce an abundant array of hydrolytic enzymes and toxins that have the potential to degrade host tissue, contributing to existing pathologic changes and further compromising intestinal integrity. Clostridia likely derive nutritional benefit from the damaged tissue and subsequent fluid accumulation. s To gain further advantage, C. difficile is known to produce inhibitory metabolic products, including p-cresol, ammonia, and volatile fatty acids such as isocaproic acid.· Metabolic byproducts from other clostridia are also likely to enhance their own competitiveness in this milieu. The resulting alteration in microflora may also result in an increase in coliform count and more endotoxin production with resultant weakening of the mucosal barrier against endotoxin absorption as well as the direct tissue damage caused by clostridial factors. Antibiotic treatment is emerging as a factor commonly associated with acute enterocolitis. In some cases, it has been considered solely responsible for inducing acute colitis. 24 The risk of inducing enterocolitis is greatly increased by the use of certain antibiotics, especially when administered orally, in combination
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with the predisposing factors previously described and the presence of toxigenic Clostridium spp. Antibiotic-associated enterocolitis generally occurs within the first week after treatment is initiated, with the onset ranging from 1 to 8 days 2, 12, 24 Antimicrobials incriminated in enterocolitis include the f3-lactams (ampicillin, penicillin, and ceftiofur), erythromycin, metronidazole, aminoglycosides, and trimethoprim-sulfamethoxazole,lhlK The accidental ingestion of small amounts of erythromycin by mares during the treatment of their foals with this antibiotic was considered to be responsible for the changes in their intestinal microflora resulting in the development of acute colitis with peracute or acute onset and a fatal outcome in nine cases,'- 24 Interestingly, this problem has only been described in Sweden, It was considered to be such a serious problem that veterinarians replaced erythromycin with gentamicin for the treatment of R, equi infection in foals, without any further occurrence of the problem,3 The antimicrobials with the greatest risk of being associated with enterocolitis are those whose spectrum of activity includes anaerobes, Obligate anaerobic bacteria, which are considered to be the most important group responsible for maintaining colonization resistance, are usually susceptible to lincosamides, macrolides, f3-lactams, and tetracyclines, Treatment with these antibiotics has also been associated with enterocolitis in horses, which ranged from diarrhea to acute fatal colitis,2, n 3" The anaerobes are less susceptible to potentiated sulfonamides, fluoroquinolones, and aminoglycosides; as a result, the use of these antimicrobials is less frequently associated with diarrhea, Orally administered antimicrobials and those that undergo enterohepatic circulation or excretion into the intestine (tetracyclines, Iincosamid es, macrolides, and some cephalosporins) pose a greater risk of disrupting intestinal microflora than parenterally administered antimicrobials that do not gain access to the lumen of the intestine in an active form, DIAGNOSIS
The diagnosis of clostridial enterocolitis is based not on the clinical features of the disease but rathe r on the outcome of particular microbial and microbiologic laboratory examinations, The laboratory identification of Clostridium spp or toxins in feces from horses is usually considered to have diagnostic value, but achieving a definitive diagnosis is challenging and frequently impossible,12 Because various pathogenic clostridia and toxin types have been demonstrated in animals without signs of clostridial disease, such findings per se are not necessarily significant. The disruption of colonization resistance may allow the overgrowth of a toxigenic ClostridiulI1 sp without it necessarily contributing to the pathogenesis of disease, In most cases, microbiologic findings should be considered as providing supportive or presumptive evidence of a diagnosis. Confirmation of a diagnosis is most readily achieved when positive microbiologic findings can be correlated with lesions in the intestine that are consistent with the expected pathologic responses caused by the identified toxin or clostridial toxin type. For this reason, a definitive diagnosis of clostridial enterocolitis is frequently made only on postmortem examination ," Clostridium perfringens Diagnosis
The conventional diagnosis of C perfrillgclIs infection is based on isolation of the organism and demonstration of toxins in feces or intestinal contents, 12
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Some commensal carriage of C. pClfril1gells can be expected, howevel~ such that detection of organisms without evaluating their numbers or toxin production is of little diagnostic or prognostic value.,e " Most clinical laboratories expect to isolate C. perfrillgclIs using routine anaerobic media. Considerable variability exists among the culture methods used. On comparison of fiv e methods for culturing feces for C. pcrfril1gclls, the most sensitive method only detected 74% of the positive samples. In all cases, C. pClji-ingclls was d etected in at least one sample from which it was not isolated by any other method. It is thus necessary to use several methods when atte mpting cultivation of C. perti'illge1ls if the epidemiologic association between C. jiC/ji"ingeI15 and enterocolitis is to be accurately assessed and the predictive value of cultures for clinical diagnosis is to be validated." n Toxin detection and id entification are recommended diagnostic procedures to be used in conjunction with attempted isolation of C. jiclfrillgcl1s.' 7 Confirmatory evidence that toxins were produced by C. jicrfrillge1l s in vivo is based on identifying them in feces or intestinal contents . Toxin ne utrali za tion tests for detecting the major lethal toxins are not readily available, however. A few immunoassays for toxin detection are available primarily for detecting enterotoxin. ' The reverse passive latex (lgglutination assay (Oxoid PET-RPLA kit; Oxoid Inc., Ogdensberg, NY) is prone to giving a high rate of false-positive results. It is of little diagnostic value, because less than lO'Yo of the isolates from horses that tested positive by reverse passive latex agglutination assay were positive for the enterotoxin gene by PCR assay. II. " PCR multiplex assays for determining the toxin genotype of C. pClfrillgclls in isolates or fecal samples are currently the preferred diagnostic approach. ' ''·"" These ultrasensitive assays are unlikely to provide false-negative results unless some unrecognized toxin is present such as the recent discovery of the [32 toxin. It should be emphasized that positive findings do not diffe rentiate between the mere presence of the toxin genotype and the expression of toxin at biologically significant levels in the intestine. Results must be evaluated in light of other diagnosti c findings. Clostridium difficile Diagnosis
Diagnosis of C difficile infection requires isolation of the bacterium from feces or demonstration of cytotoxin in feces.' C. difficilc shedding by asymptomatic horses and foals at leve ls that are readily d etected by culture procedures is apparently rare. Because nontoxige nic strains are occasionally identified, toxin detection or testing of isola tes for toxigenicity is recommend ed. C. difficile is difficult to isolate, because culture techniques that e nhance germination of spores but prevent overgrowth by other enteric bacteria must be used. Media containing ingredients that enhance isolation and recognition of C. difficilc in hum an fecal samples are commercially available"' ; however, the optimum formulation of media for isolation of C. difficile from horses has not been critically evaluated at this time. In some cases, horses with acute colitis have been found to be culturepositive but not cytotoxin-positive until 1 or 2 days later2lS. " Because the detection of C. difficile infection in
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detection based on PCR amplification of the genes for toxins can be applied directly to fecal samples or isolated bacteria." 7, 22 These methods provide an increased sensitivity of between 10- and 100-fold over tissue culture detection of toxin. The direct PCR method may be most useful in elucidating the epidemiology of C. difficiZe infection by allowing detection of extremely low numbers of bacteria in specimens and the environment. Epidemiologic tracking of sources of infection can be accomplished by molecular analytic methods using a wide variety of markers that have identified over 400 unique types. 27 In addition, a variety of strains can be differentiated by antibiograms 22 and by serotyping based on cell wall antigens?
Sample Collection and Handling
As is the case with other bacterial infections, proper selection, collection, and transportation of clinical specimens are extremely important in optimizing the chances of laboratory diagnosis of clostridial enterocolitis.] For optimal results, a freshly collected fecal sample (ideally 20-30 mL) that has not become contaminated with environmental dust or soil is the preferred sample for culture or toxin assay. Swab specimens are inadequate for toxin assay, because the volume of fecal material obtained is too small. Swabs may be acceptable for epidemiologic investigations using sensitive assays (e.g., gene probes) or enrichment culture, where the objective is to determine carrier prevalence rather than to provide quantitative results that may aid in the interpretation of clinical significance and enable confirmation of a diagnosis. To optimize bacterial isolation or detection of toxins, samples should be processed within 2 hours of collection. Sporulation rates, survival of vegetative clostridial cells, and stability of fecal toxins vary greatly among the clostridia; thus, recommendations for acceptable handling of a specimen for one assay are not necessarily optimal for all possible assays. Although spores survive for several days in refrigerated samples, a large decrease in the number of viable vegetative cells of clostridia can be expected. Prolonged storage of fecal samples at ambient or refrigerated temperatures is also likely to lead to denaturation of clostridial toxins. Freezing samples at - 70°C is thus routinely recommended for optimal long-term preservation of samples for culture and toxin detection. There is a critical need for comprehensive evaluation of the diagnostic methods used for detection of clostridial enterocolitis in horses that would establish the optimum number and periodicity of fecal sampling for clostridial culture and toxin detection.]7 Although little clinically useful information is gained by repeating assays for C. difficile within 7 days in humans/, the same recommendation does not seem to apply to horses. Multiple samples must be collected, because the nonhomogeneous nature and volume of equine intestinal contents and feces may not permit ready detection of the organism. Early in the course of infection, clostridia may be more closely associated with mucosal surfaces and may not be found in feces. Feces can continue to be normal in appearance even though the animal may be exhibiting peracute signs of enterocolitis. The diagnostic challenge of detecting and associating C. difficile with equine clostridial enterocolitis is well illustrated by reports where the organism or its toxins were not detected until late in the course of the disease after up to 5 days of daily serial sampling. 45, ,. Daily fecal samples should thus be collected and submitted to the laboratory until the clinical signs of disease begin resolving or a positive result is obtained.
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TREATMENT
The fatality rate for clostridial enterocolitis is highly variable (10%-70%) among treated horses and depends on the virulence of the pathogen, susceptibility of the host, and aggressiveness of the therapy." Horses with acute enterocolitis may require a combination of fluid therapy, nonsteroidal anti-inflammatory drugs, antidiarrheal agents, antimicrobial agents, dietary management, and other supportive care as previously reported in the literature. 13 This discussion is limited to treatment options for the clostridial component of the acute enterocolitis syndrome. Antimicrobial Treatment
Antimicrobial drugs with activity against clostridial organisms that are appropriate for use in the horse include bacitracin, metronidazole, and vancomycin. Oral bacitracin has been recommended based on the treatment and prevention of idiopathic colitis induced in horses, which is presumably caused by unidentified Clostridium Spp.S1l In vitro determination of the antimicrobial susceptibilities of 105 C. difftcile isolates from horses revealed that all were resistant to greater than 1024 fLg of bacitracin per milliliter.co It seems that bacitracin is not a logical first-line drug of choice for treating clostridial enterocolitis if C. difficile is possibly involved. 54 Successful treatment of equine clostridial enterocolitis with metronidazole has been reported. Statistically, metronidazole improved the survival rate for acute nosocomial enterocolitis presumed to be caused by clostridia. 3" A 10-day course of metronidazole treatment was associated with clinical improvement and full recovery in a mare with acute enterocolitis attributed to infection with C. difficile and C. perfringens. 1h Up to 40% of C. difftcile isolates from horses at one veterinary hospital were classified as resistant to metronidazole, however, based on in vitro antimicrobial susceptibility testing. 2b , 35 Insufficient data are available from clinical trials in horses to determine the efficacy or complications of metronidazole treatment or to provide a definitive antimicrobial treatment recommendation for clostridial enterocolitis, There are no statistically significant differences in the response or relapse rates for oral vancomycin compared with oral metronidazole treatment in human patients. 55 Nevertheless, metronidazole is considered to be the first-line drug of choice, because vancomycin is much more expensive and poses a greater risk of selecting glycopeptide resistance in other pathogens such as enterococciY Despite appropriate antimicrobial therapy, approximately 15°/r, to 25% of human patients experience relapses of C. difficile enterocolitis once treatment is discontinued. At least half of these putative relapses are, in fact, cases of reinfection with a different strain of C. difficile. 55 Because the vegetative cells of clostridia are susceptible to antimicrobial drugs, the presence of spores may be responsible for an apparent lack of response to treatment. This emphasizes the fact that susceptibility to new clostridial strains with reinfection due to impaired colonization resistance continues until the underlying predisposing deficits are corrected. PREVENTION Immunization
Vaccines with proven safety and efficacy to reduce the risk of clostridial enterocolitis have not been developed for use in horses. C. perfringens type C
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and D toxoid developed for ruminants has been administered to horses, but safety and efficacy data are not available.'" '2 There are no vaccines against C. difficile. The frequency of relapses and chronic C. difficilc infections in humans suggests that the protective value of convalescent immunity may be limited. Lack of animal models and the inability to consistently reproduce disease by oral administration of organisms or toxins are major factors limiting efficacy testing of proposed vaccines. One promising approach to immunotherapy has been the production of an anti-C. difficile bovine immunoglobulin concentrate for oral administration, which is reported to inhibit cytotoxicity and enterotoxicity.55
Biotherapy
Biotherapy aims to restore the commensal intestinal micro flora and thus optimize colonization resistance against Clostridium spp. In 15'Yo to 25% of human patients, cessation of antimicrobial therapy is all that was required to re-establish colonization resistance." Although a variety of probiotics (bacterial and yeast products) have been administered to humans and evaluated in various laboratory animal models and in vitro systems, efficacy data in horses are lacking. Commercial preparations of Saccharomyces boulardii have been shown to have significant therapeutic and prophylactic potential in humans for reducing diarrhea and recurrences associated with C. difficile infection.OJ 00 S. lJOulardii was shown to prevent binding of C. difficile enterotoxin in a rat model. It should be noted that S. cerevisiae (brewer's yeast) is distinct from S. boulardii and should not be considered equivalent to a commercial preparation of S. boulardii. A combination of Lactobacillus and xylitol has also been shown to have a protective effect, possibly by inhibiting the adhesion and growth of C. difficilc. 4 ()
SUMMARY
Equine clostridial enterocolitis is being recognized with increasing frequency. It has been identified in foals with diarrhea, antibiotic-associated enterocolitis, or nosocomial enterocolitis. The sporadic occurrence of clostridial enterocolitis, the variety of types of clostridia involved, and the difficulty of experimentally reproducing the disease suggest that it is a poorly defined multifactorial syndrome. The risk factors associated with susceptibility to colonization and progressive infection are largely based on anecdotal observations and extrapolation from human studies. Quantitative studies are needed to decipher the complex interactions between host and indigenous microflora that provide for and maintain a healthy colonization resistance environment. It seems that such studies might be more beneficial in furthering our understanding of the pathogenesis of clostridial enterocolitis than attempting to implicate another agent or toxin as the sole cause of the disease in equids. Treatment protocols that interrupt the pathogenesis of the disease need to be devised and critically evaluated to complement the present protocols emphasizing supportive care. Perhaps it is time to consider clostridial enterocolitis as yet another consequence of the use of antimicrobials analogous to the selective pressures that result in the emergence of multiple drug-resistant pathogens.
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49. Staempfli HR, Prescott JF, Brash ML: Lincomycin-induced severe colitis in ponies: Association with Clostridium clldl/veris. Ca n J Vet Res 56:168- 169, 1992 50. Staempfli HR, Prescott JF, Carman RJ, et al: Use of bacitracin in the prevention and treatment of experimentally-induced idiopathic colitis in horses. Can J Vet Res 56:233- 236, 1992 51. Teale C), Naylor RD: ClostridiulIl dijficile infection in a horse. Vet Rec 142:47, 1998 52. Traub-Dargatz JL, Jon es RL: Clostridia associated enterocolitis in adult horses and foals. Vet Clin North Am Equine Pract 9:411-421,1993 53. Tschirdewahn B, Notermans S, Werna rs K, et al: The presence of enterotoxigenic Clostridiu111 perfrillgclls strains in faeces of various animals. lnt J Food Microbiol 14:175178,1992 54. Weese JS, Parsons A, Staempfli HR: Association of ClostridiulIl difficile with enterocolitis and lactose intolerance in a foal. JAVMA 214:229-232, 1999 55. Wilcox MH: Treatment of Clostridiu111 difficile infection. J Antimicrob Chemother 41(suppl C):41-46, 1998
Address repril1t requests to Robert L. Jones, DVM, PhD Department of Microbiology Colorado State University Fort Collins, CO 80523 e-mail: [email protected]