S2 - O - 04 EVALUATION OF A LATEX AGGLUTINATION TEST FOR THE IDENTIFICATION OF CLOSTRIDIUM DIFFICILE OF PORCINE ORIGIN Introduction Clostridium difficile-associated disease (CDAD) in piglets less than 1 week old is considered emerging and <strong>of</strong> great importance for its direct effects on piglet performance. Although no direct evidence <strong>of</strong> animal-to-human transmission, isolation and accurate identification <strong>of</strong> CD strains are crucial for both diagnostics and epidemiology <strong>of</strong> CDAD, due to the significant overlap between strains implicated in CD infection in humans and animals. A definitive diagnostic <strong>of</strong> CD infection is usually achieved by combining toxin testing <strong>of</strong> stool specimens, along with isolation and further toxin production test by CD isolates. Jaime Maldonado 1 , Laura Valls 1 1 Hipra, Diagnostic Center, Girona, Spain Clostridium difficile, latex, agglutination In addition to the colony morphology and growth features in selective culture media, definitive identification <strong>of</strong> CD in pure cultures is <strong>of</strong>ten accomplished by biochemical pr<strong>of</strong>iling, and by PCR amplification <strong>of</strong> CD-specific or toxin genes. Commercial latex agglutination test (LAT) for presumptive identification <strong>of</strong> CD in stools, broth cultures and solid media are available as an alternative to costly and labour-intensive assay. They are rapid and simple assays for the early identification <strong>of</strong> CD. In this study we aimed to evaluate the usefulness <strong>of</strong> a LAT kit for the presumptive identification <strong>of</strong> CD strains <strong>of</strong> porcine origin. Materials & methods The C. difficile Test Kit (Oxoid, Hampshire, UK) was evaluated using three groups <strong>of</strong> bacteria: 1) The test was validated with five well identified CD strains <strong>of</strong> porcine origin from previous studies (1), with known toxin and ribotype pr<strong>of</strong>iles; 2) Further evaluation was carried out with 41 CD field isolates coming from suckling piglets suffering from diarrhoea in the 1 st week <strong>of</strong> age. They came from the same number <strong>of</strong> epidemiologically unrelated pig breeding units, and were collected in the period 2008-2011. Bacterial culture and identification were performed as previously described (1). Definitive identification to species level was carried out using the API Rapid ID 32 A kit (BioMerieux, Marcy l'Etoile, France); 3) Cross-reactivity in the LAT kit was determined using reference and collection strains <strong>of</strong> swine Escherichia coli (n=4), Clostridium perfringens types A, B, C, D and E (n=5), Salmonella typhimurium (n=1), Proteus mirabilis (n=1) and Clostridium novyi (n=1). The LAT kit was performed as per manufacturer’s instructions, with all three groups <strong>of</strong> bacteria involved in the study. Agglutination reactions were scored as negative (0), mild (1+), moderate (2+), or strong (3+). Results When LAT was carried out with well characterized CD antigens <strong>of</strong> porcine origin, it correctly identified all five bacteria with a strong (3+) agglutination reaction. Similarly, all 41 field CD strains identified by the API system with excellent (24,4%) or good (75.6%) identification scores, also tested positive with 1+ (14.6%), 2+ (29.3%), or 3+ (56.1%) agglutination reactions. No correlation between the two scores was observed. Nevertheless, with respect to cross-reactivity, two strains <strong>of</strong> C. perfringens (Types A [3+] and E [1+]), one strain <strong>of</strong> E. coli (3+), and the swine pathogen C. novyi (2+) tested positive in the LAT, and hence were incorrectly identified as being CD. Figure 1: Latex agglutination test: 1. Swine C. difficile strong reaction (3+); 2-4. Cross-reactivity <strong>of</strong> swine C. novyi (2+), C. perfringens type A (3+) and E. coli (3+), respectively; 5. Negative control <strong>of</strong> the test. 6. Positive control <strong>of</strong> the test. Discussion & conclusion Taking together, the results obtained in this study indicates that the LAT kit has a good ability to identify CD, since none <strong>of</strong> the collection or field CD strains tested negative. However, the fact that three bacterial species that are part <strong>of</strong> the pig intestinal flora have tested positive, indicates that positive results using this kit should be interpreted with caution. In fact, the kit manufacturer warns <strong>of</strong> the need to confirm all positive results, which is evidenced in this study. Considering that a wide variety <strong>of</strong> CD strains were tested by LAT in the present study, it is reasonable to assume that negative agglutination test results obtained with the evaluated kit, under the described conditions (pure cultures on solid media), may correspond to true negative. Consequently, the LAT kit described could eventually be used to test porcine bacterial cultures suspicious <strong>of</strong> being CD; positive results must be confirmed by a more specific assay, and negative results should be coupled with growth features and case history. Although CDAD is considered a major threat for the swine industry, and despite the obvious similarities between the CD strains affecting humans and domestic animals, existing diagnostic tools have not been evaluated in depth using clinical samples <strong>of</strong> porcine origin. It would be necessary to do so, with those test intended for toxin detection in stools and molecular detection <strong>of</strong> toxin genes, in order to improve the current diagnostic approach to CD-related neonatal diarrhoea in swine. Acknowledgements We thank Carmen Sánchez and Lidia López who collected and identified bacterial strains during the study. References 1. Alvarez-Perez S, Blanco JL, Bouza E, Alba P, Gibert X, Maldonado J, Garcia. (2009). Prevalence <strong>of</strong> Clostridium difficile in diarrhoeic and nondiarrhoeic piglets. Vet. Microbiol, 137, 302-5.
Oral presentations “Emerging, re-emerging and wildlife diseases – diagnostic possibilities” (3 rd session)
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