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   Swine Health and Production  —    Volume 8, Number 4 185 C   Diagnostic Notes Infection of neonatal swine with Clostridium difficile  J. Glenn Songer, MA, PhD; Karen W. Post, DVM, MS; David J. Larson, DVM, MS; B. Helen Jost, PhD; Robert D. Glock, DVM, PhD Summary Clostridium difficile is an established cause of antibiotic-associated diarrhea and pseu- domembranous colitis in humans and do- mestic and laboratory animals. Diagnostic findings support a role for C. difficile in neonatal enteritis of pigs. A typical case  will occur in a piglet 1  –  7 days of age, with diarrhea beginning soon after birth. Pathol- ogy includes moderate to severe mesoco- lonic edema, sometimes accompanied by hydrothorax and/or ascites, with scattered foci of suppuration in the colonic lamina propria and accumulation of neutrophils in the mesocolon. Exudation of neutrophils and fibrin into the lumen gives rise to so - called “volcano” lesions. Cultures of affec- ted tissues commonly yield heavy growth of C. difficile , and toxin testing almost in-  variably reveals the presence of toxins A and B. Treatment with antimicrobials and use of probiotics has yielded mainly unsat- isfactory results, but bacitracin methylene disalicylate may be useful for prevention.  The lack of commercially available immu- noprophylactic products may cause pro- ducers to turn to autogenous bacterin/tox- oids, but their effectiveness is uncertain. Keywords: Clostridium difficile , neonatal pigs, mesocolonic edema, bacitracin meth- ylene disalicylate lostridium difficile is a gram-posi- tive to gram-variable, spore-form- ing anaerobe which is found widely in soil, water, and the intestinal tract of  various mammals, birds, and reptiles. It is an established cause of antibiotic-associated diarrhea and pseudomembranous colitis in humans, enterocolitis in foals, nosocomial diarrhea and typhlocolitis in adult horses, and typhlitis in adult hamsters (Table 1). Germination of C. difficile spores in the large intestine is usually uneventful, but if the normal flora has not been established or has been disrupted in some way, C. difficile may establish, multiply, and pro- duce toxins. Infections often occur as a result of exposure to antimicrobials, but dietary changes may also potentiate infection. Clostridium difficile colonizes young ani- mals and human infants, but is rapidly dis- place d as the flora matures. Despite the presence of high concentrations of stool cytotoxin (toxin B) in human infants, they are resistant to clinical manifestations of disease, apparently because neonatal enter- ocytes lack toxin receptors. Clinical manifestations and pathologic lesions  Although it remains difficult to reproduce the disease, diagnostic findings to date sup - port a working hypothesis that C. difficile is capable of producing important pathologic changes in the intestines of piglets. Infec- tion of pigs with C. difficile  was apparently first confirmed by cultural isolation in 1980 1 in gnotobiotic pigs experimentally infected with  Brachyspira hyodysenteriae and accidentally exposed to C. difficile . These pigs excreted mucoid feces containing specks of blood and became dehydrated. Inoculation with C. difficile alone produced similar signs and lesions; 1    10 7  –  1    10 8 colony-forming units (CFU) of C. difficile  were recovered per g of intestinal content, and toxin could be detected at a dilution    l0 -4 . Natural infection was reported in 8-  week-old conventional pigs, from which luxuriant growth of C. difficile  was ob- tained. 2  A diphtheritic layer covered the colonic mucosa, and stunting of ileal villi  was also observed, with evidence of regen- erative activity throughout the small intestine.  An excellent recent description of an out- break of enteritis associated with C. difficile is, for practical purposes, the only such ac- count available. 3 Pigs averaging 5 days of age displayed dyspnea, mild abdominal distension, scrotal edema, and occasional diarrhea. Ascites (> 50 mL), conspicuous edema of the ascending mesocolon, hy- drothorax, and precipitation of urates in the kidney were common. Microscopic examination revealed severe submucosal and mesocolonic edema in the ascending colon, with multifocal exudation of mucus, fibrin, and neutrophil aggregates. Both C. difficile and its toxins were detected, and the latter is considered pathognomonic for C. difficile -associated disease. In another outbreak confirmed by toxin detection (Waters E, personal communication, 1999), there was no evidence of predisposi- tion by use of antimicrobials, but two were “high - health” herds, and may have been immunologically naïve. Mortality reached 50% in suckling piglets. In Quebec, out- break-associated mortality of 16% was mainly the result of respiratory distress (due to ascites and severe mesocolonic edema). Case reports Recent cases from the records of the Rollins Animal Disease Diagnostic Labora- tory reveal aspects of C. difficile -associated enteritis that are useful in diagnosis and development of control strategies. Case 1  Three piglets were submitted with a history of diarrhea beginning at 2  –  3 days of age. Herd morbidity was 7%  –  8%. Sows had  JGS, BHJ, RDG: Department of Veterinary Science and Microbiology, University of Arizona,  Tucson, AZ 85721; email: gsonger@u.arizona.edu ; KWP: Rollins Animal Disease Diagnostic Laboratory; DJL: Iowa Veterinary Diagnostic Laboratory This diagnostic note has not been peer refereed. This article is available online at http://www.aasp.org/shap.html . Songer JG, Post KW, Larson DJ, et al. Infection of neonatal swine with Clostridium difficile . Swine  Health Prod. 2000;8(4):185  –  189. received a commercial  Escherichia coli  vac- cine. At necropsy, pigs had dark yellow di- arrhea, scant contents in the small intes- tines, and marked edema of the mesocolon. Large intestinal contents were yellow to dark yellow. One pig had milk in the stom- ach and two did not. Microscopic lesions  186  Swine Health and Production  —   July and August, 2000 included limited multifocal suppurative infiltrate in the large intestine. The colonic mucosa was generally intact with the ex- ception of some very focal erosions with loss of superficial epithelium. The submu - cosa and serosa were edematous and there  was infiltration of mixed inflammatory cells, including some mononuclear cells and neutrophils. Large numbers of small rod-shaped bacteria were also observed in the lumen and the mesocolon was edema- tous. No signific ant lesions were seen in small intestine, liver, or mesenteric lymph nodes.  Aerobic bacteriologic culture of small intes- tine yielded moderate growth of a non- hemolytic  E. coli and anaerobic cultures yielded moderate numbers of C. perfringens type A, which was nonenterotoxigenic and did not produce  2 toxin. Neither latex agglutination tests nor direct electron mi- croscopic examination revealed the pres- ence of rotavirus, and examination of fro- zen sections by a fluorescent antibody test  was negative for transmissible gastroenteri- tis virus (TGEV). Anaerobic cultures of the colon yielded moderate numbers of C. difficile and toxins A and B were detected in large intestinal contents examined by an enzyme immunoassay. Case 2  Three piglets, 4 days old, were submitted  with a history of diarrhea beginning at 3  –  4 days of age. Mortality was low but affected pigs were usually stunted. Necropsy revealed stomachs filled with milk, a dehydrated subcutis, and a small intestine which was flaccid and pale. Co - lonic contents were yellow and fluid -to- pasty in consistency, and the mesentery was edematous.  There were some damaged villi in the small intestine, and jejunal villus tips were ede- matous. There was an accumulation of fib - rin and some infiltration of mixed inflam - matory cells, predominantly neutrophils, in the lamina propria of the apical portion of the villi. The colonic mucosa was generally intact with no significant lesions. There  were a few scattered inflammatory cells, including some neutrophils, in the submu- cosa. The serosa and mesentery were ede- matous and infiltrated with a scattering of inflammatory cells. Small lymph nodes in the mesentery appeared to be somewhat hyperplastic, and macrophages in the pe- riphery of the nodes contained some he- mosiderin. Moderate numbers of large rod- shaped bacteria were found in the colonic lumen. Bacteriologic culture of the small intestine revealed moderate numbers of non- hemolytic  E. coli and low to moderate numbers of C. perfringens of two colony types. These genotyped as nonenterotoxi- genic,  2 toxin-negative type A and nonen- terotoxigenic,  2 toxin-producing type C.  As in Case 1, tests for rotavirus and TGEV  were negative. Heavy growth of C. difficile  was obtained from the large intestine, and intestinal contents contained toxins A and B as determined by enzyme immunoassay. Case 3  Three 10-day-old pigs were submitted with a history of diarrhea. The herd morbidity  was 10%, and the case fatality rate aver- aged 35%. Diarrhea began at about day 10 of age. Processing of pigs had included injection with ceftiofur sodium, and the same antimicrobial had been used for treat- ment with some improvement noted. Necropsy revealed stomachs well filled with milk curd. The small intestine was con- gested and flaccid, and the spiral colon was edematous. All pigs had pasty yellow feces. Microscopic examination of sections of small intestine revealed congestion of the submucosa and mucosa but no other signi- ficant lesions. The colonic mucosa was his - tologically normal with some areas of mild to moderate congestion. Separation of cells in the serosa indicated edema. These areas contained a scattering of mononuclear inflammatory cells with an occasional neu - trophil. There were heavy populations of large rod-shaped bacteria in the lumen of the small and large intestine. Bacteriologic culture of small intestine yielded heavy growth of  -hemolytic  E. coli,  which genotyping revealed to contain genes for the K88 pilus and toxins STb and LT. A few colonies of C. perfringens  were isolated from the small intestine, and these genotyped as nonenterotoxigenic,  2 toxin-nonproducing type A. As in Cases 1 and 2, tests for rotavirus and TGEV were negative. Moderate-to-heavy growth of C. difficile  was obtained from the colon, and intestinal contents contained toxins A and B, as determined by enzyme immunoassay. Based upon these cases and others, includ- ing those described in the literature, the case definition would typically include pig  - lets 1  –  7 days of age, presenting with a his- tory of diarrhea shortly after birth (Table 2). Litters from both gilts and sows are affected, and respiratory distress and de- creased survival rates are common. Gross pathology includes moderate to severe edema of the mesocolon (Figure 1), often accompanied by hydrothorax and/or as- cites. Large intestines are frequently filled  with pasty to watery yellowish feces. Mi- croscopic lesions in the colon consist of scattered foci of suppuration in the lamina propria, with additional accumulation of neutrophils in the mesocolon. A common lesion is edema involving the colonic serosa Table 1:Diseasesassociated with Clostridiumdifficile infection Species Human Condition(s)  Antibiotic-associated diarrhea Pseudomembranous colitis Toxic megacolon Septicemia Myonecrosis Laboratory rodents Typhlitis in hamsters  Antibiotic-associated diarrhea in mice, rabbits, and guinea pigs Horses Hemorrhagic necrotizing enterocolitis in neonatal foals Nosocomial diarrhea and typhlitis in adult horses Dogs Chronic diarrhea Ratites Enterotoxemia in ostriches Pigs Neonatal necrotizing colitis   Swine Health and Production  —    Volume 8, Number 4 187 and mesentery (Figure 2), and there are infiltrations of mononuclear inflammatory cells and neutrophils in the edematous ar- eas. There may be segmental erosion of the mucosal epithelial surface of the colon. Exudation of neutrophils and fibrin from these infl amed mucosal segments into the lumen give rise to so-called “volcano” le- sions (Figure 3). Gram stains of smears or sections may reveal large numbers of gram- positive rods. There are usually no remark- able lesions in the small intestines of pigs  with uncomplicated C. difficile infection.  Villous blunting, congestion of mucosal  vessels, atonicity, and pale contents in the small intestine are often accompanied by concurrent infection with C. perfringens or  E. coli ; the possible presence of these or  viral agents should be documented.  At the time of processing, neonatal pigs are commonly treated with penicillins or ceph- alosporins for prophylaxis. The sensitivity of the developing gut fl ora and the possible resistance of C. difficile to these antimicro- bials may be potentiating factors in the incidence of C. difficile -associated entero- colitis. Currently available data warrant no firm conclusions, but anecdotal evidence suggests increased incidence on those pre- mises where such processing is practiced.  At the present time, we do not have access to data obtained via processing of a ran- dom or representative sample from the population at risk. Thus, accurate estimates of incidence and prevalence are not avail- able. However, widespread occurrence of C. difficile -associated disease in pigs is implied by its diagnosis in laboratories throughout swine-producing areas of the United States. Data from one of these labo- ratories suggests that annual incidence peaks in January through March, and that cases involving C. difficile as an etiologic agent represent approximately 52.8% of the total neonatal pig enteritis accessions; more than 36% of these cases involved C. difficile as the only pathogen of interest. Subjectively, this may place the importance of C. difficile -associated disease above that of  E. coli , C. perfringens type A, and viral agents in at least some parts of the United States. Bacteriologic culture and toxin testing Cultures of affected tissues (Table 3) com- monly yield heavy growth of C. difficile, and toxin testing by neutralization in Chi- nese hamster ovary (CHO) cells or com- mercial enzyme immunoassay almost in-  variably reveal the presence of toxins A and B.   Results of our preliminary studies sug- gest a 93.7% correlation between clinical, pathologic, and bacteriologic diagnosis, on the one hand, and toxin detection on the other. Toxin detection in rectal swabs is also an acceptable protocol, in that results are 92.8% correlated with those from ex- amination of intestinal contents. A proto- col for the CHO cell assay, a list of manu- facturers of enzyme immunoassays, and procedures for bacteriologic culture are available. 4  Pathogenesis  Virulence attributes may include pili, cap- sule, and degradative enzymes, but produc- tion of toxins is essential. Toxins A (307 kDa) and B (270 kDa) of C. difficile are the largest bacterial toxins described to date, and are members of the family of so- called “large clostridial toxins.” The former is an enterotoxin  which causes fluid accu - mulation in the intestine, and the latter is a cytotoxin which is highly cytopathic for cultured cells. Both toxins are internalized by target cells and disrupt the cytoskeleton by enzymatic attack on intracellular targets. Cessation of protein synthesis and cell divi- sion is followed by exfoliation of entero- cytes. Degranulation of mucosal mast cells and release of inflammatory mediators causes an influx of granulocytes,  resulting in substantial tissue damage. 5  In the only study addressing pathogenesis of C. difficile infection in pigs, crude prepa- rations of toxins A and B had minimal ef- fect when administered into colonic loops of pigs. 6 However, inadequate toxin con- centration in the inoculum, dilution of toxin in the colonic segment, or interfer- ence with toxin action by flora or other   Table 2:Case definition for Clostridiumdifficile -associated disease in neonatal pigs History and clinical signs Features Piglets (from gilts and sows) 1  – 7 days of age, with diarrhea beginning shortly after birth; loss of condition, stunting of survivors; respiratory distress, decreased survival rates common Morbidity 10%  – 90%, averaging 20% Case fatality rate up to 50%, averaging 20% Possible association with administration of penicillins or cephalosporins at processing Gross pathology Moderate to severe mesocolonic edema; hydrothorax and/or ascites occasional; pasty to watery yellowish colonic contents Microscopic pathology Scattered suppurative foci in colonic lamina propria; neutrophilic infiltrate in mesocolon; segmental erosion of colonic mucosal epithelium; volcano lesions (neutrophil and fibrin exudation into colonic lumen); large rods, sometimes with spores, on mucosal surface, in lumen No remarkable lesions in small intestine Bacteriology and toxin testing Moderate to heavy growth of C.difficile Presence of toxins A and B  188  Swine Health and Production  —   July and August, 2000 Gut contents or rectal swabs (in appropriate anaerobic transport medium* if shipment requires >24 h), shipped on wet ice * See reference 4 materials may have influenced the results.  There is clearly a need to reexamine the effect of toxins A and B on the pig intes- tine, using high- titer crude and purified toxins and cultures of C. difficile . Prophylaxis and therapy Due to the relative lack of information on C.   difficile -associated enteric disease in swine, one must look to other species for prophylactic and therapeutic options. Met- ronidazole, bacitracin zinc, and vancomy- cin have been used to treat C. difficile in- fections in the horse 7,8 and the last is the antimicrobial of choice for the treatment of    typhlitis in hamsters. 9 Metronidazole and  vancomycin are not approved for use in food animals, but bacitracin methylene disalicylate (BMD ® , Alpharma; Fort Lee, New Jersey), is approved to prevent and treat enteritis due to C. perfringens in pigs. It is typically administered to pregnant fe- males at 250 g per ton of feed for 2 weeks prefarrowing and in the lactation ration at the same concentration for 3 weeks post- farrowing. 10  Anecdotal evidence in some   North Carolina outbreaks indicate that this may be effective. In some outbreaks in pigs, treatment with antimicrobials and use of probiotics has produced inconsistent, and mainly unsatisfactory, results (LaRochelle D, personal communication, 1999). Clostridium sordellii toxins are antigenically related to toxins A and B, and C. sordellii antitoxin has been administered orally to prevent enteritis in the hamster model of C.   difficile infection. 11  A nonpathogenic yeast, Saccharomyces boulardii , can suppress intestinal overgrowth by C. difficile after antimicrobial therapy. 12,13 Live, nontoxi- genic strains of C. difficile can fill niches opened in the gut of the hamster by anti- microbial therapy, as evidenced by the sub- sequent protection against challenge with Figure 1: Four-day-old piglet with gross lesions suggestive of Clostridium difficile infection, with prominent edema of the mesocolon Specimen Piglets Feces Figure 2: Microscopicappearance of the mesocolon in Clostridium difficile infection Figure 3: Suppurative focus(“volcano”lesion) in the colonic mucosa of a piglet infected with Clostridium difficile .Exudation of neutrophils into the colonic lumen isacommon finding. Table 3:Specimen selection for diagnosisof Clostridium difficile infection in neonatal pigs Comments Live, typical clinical signs, untreated Ligated loops of small intestine and colon, shipped on wet ice (not frozen)
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