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Brachyspira innocens (left), B. pilosicoli (middle) and B. hyodysenteriae (right). Photo: Bengt Ekberg/SVA
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Biochemical tests The following biochemical tests are useful for differentiation between species/types of Brachyspira: hemolysis, indole production, hippurate hydrolysis, alpha-galactosidase, and beta-glucosidase activity.
Hemolysis tested on Tryptocose Soy Agar with 5% bovine blood, is classified as weak or strong. Culturing a type strain of Brachyspira next to the field isolate on the same plate is essential in order to objectively determine the degree of hemolysis. Plates should not be older than seven days due to the risk of false positive B. hyodysenteriae reactions, as well as auto-hemolysis. Testing for indole production is done in the form of a spot test by smearing the growth from a culture onto a filter paper saturated with the indole reagent (1% p-dimethylaminocinnamaldehyde in 10% hydrochloric acid).
For the hippurate hydrolysis test the method of Rübsamen & Rübsamen (1986) is used. The reaction is deemed positive if a deep blue or purple colour developes, but negative if the solution turns light blue or remains colourless. A false-positive reaction occasionally occurrs if the bacterial inoculum contains agar. In that case, a brownish blue colour developes.
For a more detailed biochemical analysis, the API-ZYM test (API, 69280 Marcy-I´Etoile, France) may be used. This test consists of 20 microtubes, containing 19 substrates in a buffer. The first microtube serves as the control. The enzymes determined in the other tubes are: alkaline phosphatase, esterase, esterase-lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, N-acetyl-b-glucosidase, a-mannosidase and a-fucosidase. The tests are performed as described by the manufacturer, chiefly to determine a-galactosidase, a-glucosidase and b-glucosidase activity.
PCR-based methods The use of PCR directly on faeces or applied to primary cultures reduces the time needed for detecting Brachyspira spp. A number of PCR-based systems for B. hyodysenteriae and B. pilosicoli have been developed and are generally based on 16S rRNA- (Park et al., 1995; Fellström et al., 1997; Atyeo et al., 1998; La et al., 2003), 23S rRNA- (Leser et al., 1997; Barcellos et al., 2000; Thomson et al., 2001), nox- (Atyeo et al., 1999; Rohde et al., 2002; La et al., 2003) or tlyA-(Fellström et al., 2001) genes, but some methods based on undefined genes has also been described in the literature (Elder et al., 1994; Harel & Forget, 1995). A high specificity is often achieved by PCR, with sensitivity down to 10 - 100 cells per PCR reaction using pure cultures. However, PCR is hampered by inhibiting factors in faeces (Lantz et al., 2000; Jacobson et al., 2003). The sensitivity of PCR applied on faecal samples varies depending on the amount of material analysed (Elder et al., 1994; Leser et al., 1997; Atyeo et al. 1998; Thomson et al., 2001; La et al., 2003).
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