Evaluation of four different diagnostic tests to detect Clostridium difficile in piglets.

Clostridium difficile is emerging as pathogen in both humans and animals. In 2000 it was described as one of the causes of neonatal enteritis in piglets, and it is now the most common cause of neonatal diarrhea in the United States. In Europe, C. difficile infection (CDI) in both neonatal piglets and adult sows has also been reported. Diagnosis of this infection is based on detection of the bacterium C. difficile or its toxins A and B. Most detection methods, however, are only validated for diagnosing human infections. In this study three commercially available enzyme immunoassays (EIAs) and a commercial real-time-PCR (Becton, Dickinson, and Company) were evaluated by testing 172 pig fecal specimens (139 diarrheic and 33 nondiarrheic piglets). The results of each test were compared to those of cytotoxicity assays (CTAs) and toxigenic culture as the "gold standards." Compared to CTAs, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were, respectively, as follows: for real-time PCR, 91.6, 37.1, 57.6, and 82.5%; for Premier Toxins A&B (Meridian), 83.1, 31.5, 53.1, and 66.7%; for ImmunoCard Toxins A&B kit (ICTAB; Meridian), 86.6, 56.8, 66.9, and 80.7%; and for VIDAS (bioMérieux), 54.8, 92.6, 85.0, and 72.8%. Compared to toxigenic culture, the sensitivity, specificity, PPV, and NPV were, respectively, as follows: for real-time PCR, 93.0, 34.7, 50.0, and 87.5%; for Premier Toxins A&B, 80.3, 27.7, 43.8, and 66.7%; and for ICTAB, 80.0, 46.2, 52.8, and 75.4%; and for VIDAS, 56.4, 89.8, 77.5, and 76.7%. We conclude that all tests had an unacceptably low performance as a single test for the detection of C. difficile in pig herds and that a two-step algorithm is necessary, similar to that in cases of human CDI. Of all of the assays, the real-time PCR had the highest NPV compared to both reference methods and is therefore the most appropriate test to screen for the absence of C. difficile in pigs as a first step in the algorithm. The second step would be a confirmation of the positive results by toxigenic culture.

Acquisition of Clostridium difficile by piglets.

Clostridium difficile is recognized as an important cause of nosocomial diarrhoea in humans especially in association with administration of antibiotics. In pigs, C. difficile can cause neonatal enteritis and can be isolated from faeces from both diseased and healthy animals. The presented prospective study describes how soon C. difficile can be isolated from newborn piglets after normal parturition and how C. difficile spreads within a pig farm. Six sows, their farrowing crates and their litters at one farm were sampled until C. difficile was found in all piglets. Within 48 h after birth, all 71 piglets became positive for C. difficile (two piglets were already positive within 1h post partum), all sows became positive within 113 h after parturition and the farrowing crates were found intermittently positive. C. difficile could also be detected in air samples and in samples of teats of the sows. All isolates belonged to PCR ribotype 078. Twenty-one C. difficile ribotype 078 isolates, found at the farm, were further analyzed by MLVA (multiple-locus variable-number tandem repeat analysis) and belonged to one clonal complex, except one isolate. To be sure that piglets were not born already infected with C. difficile ribotype 078, 38 caesarean derived piglets were sampled immediately after surgery. All piglets tested negative at delivery and stayed negative for C. difficile ribotype 078 during the 21 days in which they were kept in sterile incubators. This study shows that C. difficile ribotype 078 spreads easily between sows, piglets and the environment. Vertical transmission of C. difficile ribotype 078 was not found and is very unlikely to occur.

High occurrence of various Clostridium difficile PCR ribotypes in pigs arriving at the slaughterhouse.

BACKGROUND: Clostridium difficile is recognized as an important cause of nosocomial diarrhoea in humans, especially in association with the administration of antibiotics. Furthermore, C. difficile can not only cause neonatal enteritis in pigs but can also be found in pigs without any clinical disease symptoms. Clostridium difficile had been found on pork samples destined for human consumption. However, little is known about the risk of food-borne transmission. OBJECTIVE: To elaborate the risk of food-borne transmission of C. difficile via pigs. ANIMALS AND METHODS: The occurrence of C. difficile was assessed in pigs arriving at a slaughterhouse in the Netherlands. Rectal faecal samples from 50 pigs originating from 10 different farms were taken just after the pigs were stunned and bled. These samples were examined using a real-time PCR (BD GeneOhm™ Cdiff Assay) combined with culturing following enrichment. RESULTS: Using real-time PCR, none of the faecal samples were found positive for C. difficile while after culturing following enrichment, 14 out of 50 samples (28%) contained C. difficile. The positive samples were derived from nine different farms and encompassed seven different PCR ribotypes (015 predominant). All isolated C. difficile strains were positive for the toxin A and B genes. CONCLUSION: These results indicate that C. difficile can be found in faecal samples obtained from pigs after they were stunned and bled in a slaughterhouse. CLINICAL IMPORTANCE: The potential risk of these findings on food-borne transmission via pigs and associated impact on human health cannot be excluded and needs further study.