Immunomagnetic separation of the intestinal spirochaetes Brachyspira pilosicoli and Brachyspira hyodysenteriae from porcine faeces

Abstract

Porcine intestinal spirochaetes are fastidious anaerobic organisms and, as a consequence, it has been necessary to develop various protocols to enhance their isolation from or detection in faeces.Immunomagnetic separation (IMS) is a method developed recently to improve separation of target cells from mixed cell suspensions. The purpose of the present study was to compare the relative sensitivity of IMS for isolation of Brachyspira pilosicoli and Brachyspira hyodysenteriae with current routine diagnostic methods (culture on selective media and PCR) for detection of these microorganisms in pig faeces. Neither direct nor indirect IMS methods enhanced the sensitivity of detection of either organism when performed with the recommended washings during sample processing. Performance of the IMS procedure without washing gave sensitivity at levels similar to direct culture onto selective medium. Further development of IMS techniques is required to improve isolation rates of Brachyspira species from faecal samples. INTRODUCTIONIntestinal spirochaetes are mainly members of the genus Brachyspira, which includes species commensal and pathogenic for pigs and other animals and humans. Brachyspira hyodysenteriae is recognized as the aetiological agent of swine dysentery (SD) (Taylor & Alexander, 1971) and Brachyspira pilosicoli is the aetiological agent of a disease of pigs known as intestinal spirochaetosis (Taylor et al., 1980) or porcine colonic spirochaetosis (PCS;Duhamel et al., 1998). Infection with B. pilosicoli has been reported in a wide range of hosts, including dogs (Duhamel et al., 1995), chickens and other avian species (Dwars et al., 1992;McLaren et al., 1997), guinea pigs (Vanrobaeys et al., 1998), non-human primates (Takeuchi et al., 1974) and humans (Cooper et al., 1986; Surawicz et al., 1987). As interest in the field of intestinal spirochaetes has grown, further species have been identified recently as pathogens in animal species other than pigs. For instance, Brachyspira alvinipulli has been described and shown to be pathogenic for chickens (Stanton et al., 1998) and the provisionally designated 'Serpulina canis' has been found in dogs (Duhamel et al., 1998).Porcine intestinal spirochaetes are Gram-negative, anaerobic and fastidious organisms. As a result, various culture media and incubation conditions have been assessed in order to develop the most reliable and efficient method for their isolation from faecal samples. Typically, isolation of these bacteria is achieved by culture on selective media based on trypticase soy agar or Columbia agar base supplemented with blood and multiple antibiotics to reduce the growth of other bacteria. Various antibiotic combinations have been utilized including spectinomycin (TSA-S400 medium; Songer et al., 1976), spiramycin-colistin-vancomycin (TSA-CVS medium;Jenkinson & Wingar, 1981) and spectinomycin-colistinvancomycin-spiramycin-rifampicin (BJ medium; Kunkle & Kinyon, 1988). In a comparative study of these media, BJ proved to be the most efficient in eliminating norm...

Immunomagnetic Separation and Coagglutination of Vibrio parahaemolyticus with Anti-Flagellar Protein Monoclonal Antibody

“…Roberts and Hirst (31) found that indirect IMS produced consistently better results for the detection of Mycobacterium ulcerans from heterogenous samples, increasing the sensitivity up to 10-fold. Corona-Barrera et al (13) also found that the indirect IMS method was 10-to 100-fold more sensitive for the detection of Brachyspira species in porcine feces. Based on this information, we used mAb 3-F-3 in an indirect IMS protocol by incubating an excess of the mAb (30 g/test sample) with target V. parahaemolyticus cells prior to the addition of the IMB.…”

“…IMS has been successfully used to concentrate and isolate numerous pathogens (21,26,30) and has often been used as a pre-PCR step to concentrate and separate the organism of interest from polymerase inhibitors in the sample matrix (13,18,19,31). A successful IMS method for the concentration of V. parahaemolyticus cells utilizing species-specific antibodies coupled to PCR would improve detection sensitivity and separate V. parahaemolyticus from other bacteria, eliminating interference with DNA amplification.…”

Particle Separation in Microfluidic Devices 3/4 SPLITT Fractionation and Microfluidics

“…Magnetic separation techniques have now emerged as one of the preferred methods for biological and clinical analyses because of the development of new magnetic beads with improved properties [29]. For example, immunomagnetic separation has become popular for the separation of specific macromolecules and cells from heterogeneous biological mixtures [30,31]. The manipulation of magnetic beads onchip was recently reviewed by Gijs [32].…”

Human intestinal spirochetosis – a review

“…PCR has become one of the more reliable methods, targeting the 16S rRNA, NADH-oxidase, and the 23rDNA gene specific for B. pilosicoli , B. hyodysenteriae , and S. intermedia [56], [57]. Novel techniques such as immunomagnetic separation show promise for the future [58]. Additionally, fluorescent in situ hybridization with oligonucleotide probes targeting 16S or 23S rRNA of B. aalborgi and B. pilosicoli has been reported to be applicable in formalin-fixed, paraffin-embedded intestinal biopsy specimens [59], [60].…”