Potential Role of Clostridium difficile Duodenitis Proximal Jejunitis

Toxigenic strains of Clostridium difficile were isolated from seven of seven (100%) horses with duodenitis proximal jejunitis but zero of seven controls (p < 0.0001). No other known pathogenic clostridia were isolated from either group. This study suggests that C. difficile might be an important cause of this currently idiopathic syndrome.

1. Introduction

Duodenitis proximal enteritis (DPJ) is an acute sporadic syndrome in horses characterized by inflammation and edema of the duodenum and jejunum [1]. The initiating cause of this syndrome remains unknown, however, clostridia, Salmonella spp., and mycotoxins have been suggested. White et al [2] reported that "the histopathological lesions in the small intestine of affected horses are similar to those produced by Clostridium spp. exotoxins"; however, a causal link could not be established. While typically associated with diarrhea, C. difficile can also affect the small intestine and could be a cause of DPJ. Jones et al [3] reported that severe damage occurs to the small intestine of foals suffering from natural infection or after experimental challenge with C. difficile.

The objective of this study was to further investigate the hypothesis that clostridia are involved in the pathogenesis of DPJ and to evaluate the recovery of C. difficile from horses with DPJ compared with horses with nasogastric reflux from other etiologies.

2. Materials and Methods

Nasogastric reflux was collected from seven consecutive cases of proximal enteritis and seven consecutive horses with other causes of nasogastric reflux admitted to the Ontario Veterinary College Veterinary Teaching Hospital. Samples were collected at the time of admission and handled in a blinded manner. One milliliter of reflux was inoculated into non-selective enrichment broth [a] followed by sub-culture onto blood agar and incubated anaerobically at 37° C. Clostridia were identified through colony and Gram strain morphology and identified using a biochemical system [b]. Additionally, selective enrichment culture for C. difficile consisted of inoculation of 1 ml of reflux into cycloserine-cefoxitin fructose broth enriched with 0.1% sodium taurocholate [c], incubated at 37°C for 5 days. Culture broth was inoculated onto blood agar and incubated anaerobically at 37°C for 24 - 48 h. C. difficile was identified through colony characteristics, Gram stain morphology, and production of l-proline aminopeptidase [d]. All C. difficile isolates were tested for the presence of genes encoding C. difficile toxins A, B, and CDT as previously described [4,5].

3. Results

Toxigenic strains of C. difficile were cultured from the nasogastric reflux of seven of seven (100%) horses with clinical DPJ but from none of the control samples (Fisher's exact, p < 0.001). Additionally, no other known pathogenic clostridia were isolated from either group.

Four of seven (57%) of the isolates possessed genes encoding for the production of both of the main toxins, toxins A and B, whereas three were variant strains that produced toxin B but not toxin A. Additionally, genes encoding for CDT were present in one isolate that also possessed the genes encoding for toxins A and B. Three (47%) horses with DPJ were euthanized, whereas the remaining four cases fully recovered. All cases that were euthanized were colonized by a variant (A-/B+) strain of C. difficile. Histology sections of small intestine collected from horses with fatal C. difficile-associated enterocolitis were compared with histology sections of euthanized horses with clinical DPJ. Similar histopathological changes were identified between both entities including sloughing of villus epithelium, submucosal edema, fibrinoid necrosis of vessels walls, and thrombosis of blood vessels within the submucosa, muscularis, and subserosa.

4. Discussion

DPJ is an important syndrome in the horse and can be associated with mortality, high treatment costs, and complications such as laminitis. Despite a variety of studies, an etiologic agent has not been identified. Braun et al [6] reported the isolation of C. difficile from horses with DPJ; however, no assessment of the role of this organism in disease was made. Results of this study suggest that C. difficile may be a cause of DPJ. It was surprising that C. difficile was recovered from 100% of horses with DPJ that were tested. However, DPJ and control samples were handled blindly, and all DPJ samples were serially collected samples with no pre-test suspicion of C. difficile. The apparent similarity of histological changes in horses with DPJ and C. difficile enterocolitis is further supportive.

The isolation of non-pathogenic clostridia is consistent with previous studies, which found inconsistent results and could not establish a direct cause with this disease. However, future studies specifically testing for pathogens such as C. perfringens and Salmonella should be included as part of the screening protocol.

While this study does not prove causation, isolation of C. difficile from the reflux of horses with DPJ indicates that further study of the potential role of this pathogen by means of a larger prospective and controlled study is indicated. Furthermore, associated risk factors that ultimately may favor bacterial overgrowth and toxin production also need to be studied.

Footnotes

[a] Brain heart infusion; Oxoid Inc. Nepean, Ontario, Canada.
[b] API strip test; bioMérieux Canada, Inc. , St. Laurent, Québec, Canada H4S 1P7.
[c] Cycloserine-cefoxitin fructose broth; Oxoid Inc. Nepean, Ontario, Canada.
[d] ProDisc test; Remel, Carr-Scarborough Microbiologicals, Inc. , Decatur, GA 30035.