Cite as: Silva ROS, Guedes RMC, Silva MX, et al. Evaluation of three enzyme immunoassays and toxigenic culture for diagnosis of Clostridium difficile-associated enteritis in piglets. J Swine Health Prod. 2013;21(6):300–303.
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Keywords: swine, Clostridium difficile toxins A and B, neonatal diarrhea, colitis
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Received: November 4, 2012
Accepted: March 8, 2013

C lostridium difficile is a spore-forming, anaerobic, gram-positive bacillus that has been recognized as responsible for 95% of all pseudomembranous colitis cases and most cases of antibiotic-associated diarrhea in humans.1 In veterinary medicine, this organism is considered the most important uncontrolled cause of neonatal diarrhea in pigs in some countries, including the United States and Brazil.2,3 In addition, recent studies also showed that the strains isolated from humans suffering from C difficile infection (CDI) have a high genetic relatedness to strains of animal origin,4 suggesting that CDI is a zoonosis.

For most authors, the “gold standard” for diagnosis of CDI is the cytotoxicity assay (CTA), but this test is both labor intensive and time consuming. Therefore, commercial enzyme immunoassays (EIAs) remain the most common method used for diagnosis of CDI in humans and animals.5,6 Recently, toxigenic culture has also been described as a sensitive method for human samples,7 but little is known with regard to its application for samples from domestic animals.

Despite the importance of C difficile as a swine enteropathogen and even as a potential zoonotic agent, no established guidelines are available for diagnosing CDI, and performance is unknown for most commerically available detection methods.8 In light of this fact, the aim of the present study was to compare test performances of three different EIAs and toxigenic culture to the CTA as the gold standard.

Materials and methods

Ethics approval for this study was granted by the Animal Experiments Committee of the Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

The piglets included in this study were submitted to the Veterinary School of Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, for routine diagnosis of piglet neonatal diarrhea. Live animals were euthanized and necropsied, and intestinal content was collected in sterile containers and stored at -20°C until tested up to 7 days later. A total of 73 samples from 32 farms were included in the study, with 62 samples from diarrheic piglets and 11 from non-diarrheic piglets.

The CTA for C difficile A and B toxins was performed with Vero cells (ATTC CCL 81) as described previously.9 Briefly, fecal samples were diluted 1:4 in phosphate-buffered saline (pH 7.0) and centrifuged at 3000g for 5 minutes at 4°C. The resulting supernatant was filtered through a 0.22-µm pore size filter and diluted twofold until a dilution of 1:1024 was reached. Serial dilutions and parallel samples with Clostridium sordellii antitoxin (National Institute for Biological Standards and Control, Hertfordshire, England) were added onto the Vero cell monolayers. The cells were examined after 24 hours of incubation at 37°C in a 5% CO2 incubator. A specimen was considered positive if at least 90% of the cells were rounded and the effect was neutralized by antitoxin at the same dilution in a parallel sample.

For toxigenic culture, the fecal samples were subjected to alcohol shock,10 and 50-µL aliquots were inoculated onto cycloserine-cefoxitin fructose agar (CCFA) plates (Hi-media, Mumbai, India) supplemented with 7% horse blood and 0.1% sodium taurocholate (Sigma-Aldrich Co, St Louis, Missouri). After incubation in an anaerobic chamber at 37°C for 72 hours, colonies with morphology suggestive of C difficile and a positive Gram stain were subjected to a previously described multiplex polymerase chain reaction (PCR) for a housekeeping gene (tpi), toxins A (tcdA) and B (tcdB), and binary toxin genes (cdtB).11 In addition, all toxigenic isolates in the PCR were tested by CTA for in vitro toxin production as previously described.5 Clostridium difficile ATCC 9689 was used as a control for the PCR and toxigenic culture.

Three commercial enzyme immunoassays (EIAs) for A and B toxin detection were tested: Clostridium difficile Tox A/B II (Techlab Inc, Blacksburg, Virginia), Remel ProSpecT Clostridium difficile Toxin A/B Microplate Assay (Oxoid, Hampshire, United Kingdom), and Ridascreen Clostridium difficile toxins A/B (R-Biopharm, Darmstadt, Germany). All EIAs were performed according to the manufacturers’ recommendations. The sensitivity, specificity, positive predictive value, negative predictive value, and 95% confidence intervals were calculated for each EIA and for toxigenic culture against CTA (Stata 12; StataCorp LP, College Station, Texas).

Results

The CTA was positive in 22 of the 73 samples (30.1%). Six of the positive samples were from non-diarrheic piglets. Sensitivities of the EIAs evaluated were ≤ 63.6%, and specificities were 80.3% to 98% (Table 1). Clostridium difficile was isolated from 13 of the 73 samples (18.3%), with 10 strains toxigenic by PCR. All toxigenic isolates were positive for the tcdA and tcdB genes, and one was also positive for the binary toxin gene (cdtB). One toxigenic strain isolated from a diarrheic piglet was negative for A and B toxins by CTA. All PCR-toxigenic strains were able to produce toxin in vitro. Sensitivity and specificity of toxigenic culture are provided in Table 1.

Discussion

All EIAs tested had sensitivities < 65% when used for piglet fecal samples. This undesirable EIA performance with regard to piglet fecal samples is not surprising and was previously reported for other EIAs.8,12 Some authors attributed the low specificity of EIAs for swine fecal samples to inhibitors in animal feces; however, to date, no evidence confirms this possibility.8,13 In contrast, similar to a previous report,8 false-positive results varied widely among EIAs, suggesting that the incorrect results were due to the test and not to an interfering substance in the samples. It is also interesting to note that an older version of the Techlab EIA was previously tested on porcine fecal samples, with a sensitivity of 91% and a specificity of 86% reported.14 In the present study, the new version of the Techlab test had a much lower sensitivity (59.1%), but the specificity was 98.0%.

In contrast to a previous report,8 the sensitivity of toxigenic culture in this study was low (40.9%). Unfortunately, there is no standard method for toxigenic culture of C difficile, making it difficult to compare reported results. A great variety of media have been reported, in addition to differences in isolation protocol, such as the use of alcohol shock and variations in incubation time. Accordingly, this study reports a simple isolation method that would be more applicable for diagnosis than previously reported protocols.5,8,15 In this protocol, the samples were subjected to alcohol shock, plated on CCFA supplemented with 0.1% sodium taurocholate, and incubated for 72 hours. It is well known that some C difficile strains fail to grow on CCFA because of susceptibility to one or both antibiotics used in the medium.16 In addition, the use of CCFA, even with supplemental taurocholate, may result in variable sensitivity for recovery of C difficile spores, compared with other isolation protocols, such as use of pre-enrichment broth.17 All these factors might have contributed to the low sensitivity of the toxigenic culture protocol tested and suggest that this protocol is not acceptable for diagnosis of CDI in piglets, in contrast to the previous reports for human samples.6,7

All toxigenic isolates in the present study were positive for the tcdA and tcdB genes, whereas one strain was also positive for the binary toxin gene (cdtB). It is interesting to note that all the piglet C difficile isolates that were considered to be toxigenic by PCR were also able to produce toxins A and B in vitro. This result suggests a good correlation between toxin gene detection by PCR and in vitro toxin production in porcine C difficile strains. Therefore, the toxin production test after isolation may not be necessary when A and B toxin genes have been detected. The removal of this step would save time and reduce the cost of diagnosis. Further studies with a larger number of strains are needed to confirm this hypothesis.

In the present study, one toxigenic strain isolated from a diarrheic piglet was negative for A and B toxins by CTA. Several hypotheses should be considered. First, the toxins might not have been detected by CTA, which does not exhibit 100% sensitivity.5 Second, the piglet might have been an asymptomatic carrier, and other enteropathogens might have been responsible for the diarrhea. Another possibility is that A and B toxins that had been present were degraded by fecal proteases. It should be emphasized that the time between sample collection and processing in the present study was short (only 7 days). In a previous study, A and B toxins remained detectable in piglet fecal samples for at least a month at -20°C.8 These data suggest that failure to detect A and B toxins using the EIAs in the present study was not caused by storage conditions. Some authors contend that protease activity in animal fecal specimens may cause rapid toxin degradation such that toxin may not be detectable by EIAs or CTA; to date, to the authors’ knowledge, there is no study confirming this hypothesis.13

The sensitivity and specificity of all the EIAs tested were unacceptable for testing individual piglet samples. Similar results have been reported in humans, and some studies suggest that at least a two-step algorithm is needed to reliably diagnose CDI;7 however, there is no consensus thus far on which tests should be used in each step. One possible approach is use of an EIA with a high sensitivity as the primary test, followed by CTA as the confirmatory test for positive samples. Another option is use of a high-specificity method associated with a large number of samples from each swine farm. In the present study, one of the EIAs had a specificity of 98% (95% CI, 89.7%-99.6%) for piglet samples, allowing a great degree of confidence in the positive results, with a PPV of 92.9% (95%CI, 68.5%-98.7%) and an NPV of 84.7% (95%CI, 73.5%-91.8%). Therefore, we suggest this EIA (Techlab) might be useful for screening for CDI in a herd when multiple samples are collected.

Implications

•  Under the conditions of this study, sensitivity and specificity of the three EIAs tested are unacceptable for diagnosis of C difficile in individual piglet fecal samples.

•  Use of a high-specificity EIA associated with a large number of samples from each swine farm could be used to screen for C difficile infection in a herd when multiple samples are collected.

Acknowledgments

The authors thank the following: Fundação de Amparo a Pesquisa de Minas Gerais, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, and Institutos Nacionais de Ciência e Tecnologia.

Conflict of interest

None reported.

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