Abstract Book of EAVLD2012 - eavld congress 2012

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 of great importance
for its direct effects on piglet performance. Although no direct
evidence of animal-to-human transmission, isolation and
accurate identification of CD strains are crucial for both
diagnostics and epidemiology of CDAD, due to the significant
overlap between strains implicated in CD infection in humans and
animals. A definitive diagnostic of CD infection is usually
achieved by combining toxin testing of 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 of CD in pure
cultures is often accomplished by biochemical profiling, and by
PCR amplification of CD-specific or toxin genes. Commercial
latex agglutination test (LAT) for presumptive identification of 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 of CD. In this study
we aimed to evaluate the usefulness of a LAT kit for the
presumptive identification of CD strains of porcine origin.
Materials & methods
The C. difficile Test Kit (Oxoid, Hampshire, UK) was evaluated
using three groups of bacteria: 1) The test was validated with five
well identified CD strains of porcine origin from previous studies
(1), with known toxin and ribotype profiles; 2) Further evaluation
was carried out with 41 CD field isolates coming from suckling
piglets suffering from diarrhoea in the 1 st week of age. They came
from the same number of 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 of 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 of 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 of
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 of C.
perfringens (Types A [3+] and E [1+]), one strain of 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 of swine C. novyi (2+), C.
perfringens type A (3+) and E. coli (3+), respectively; 5.
Negative control of the test. 6. Positive control of 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 of the
collection or field CD strains tested negative. However, the fact
that three bacterial species that are part of 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 of the need to confirm all positive results, which is
evidenced in this study.
Considering that a wide variety of 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 of 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 of porcine origin. It would be necessary to do so, with
those test intended for toxin detection in stools and molecular
detection of 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 of Clostridium difficile in diarrhoeic and nondiarrhoeic
piglets. Vet. Microbiol, 137, 302-5.