Extensive intestinal spirochaetosis in pigs challenged with Brachyspira pilosicoli

The weakly ß-haemolytic spirochaete Brachyspira pilosicoli is the aetiological agent of porcine intestinal spirochaetosis or colonic spirochaetosis, a transient, large-intestinal disease clinically characterized by diarrhoea with loose to watery and sometimes mucoid faeces, causing reduced growth rate and failure to thrive in pigs (Trott et al., 1996a). In addition, B. pilosicoli causes similar disease in a wide range of hosts, including humans (Taylor & Trott, 1997). Surveys from Europe have recently shown that B. pilosicoli is a prevalent pathogen among 6- to 16-week-old pigs with diarrhoea and colitis (Møller et al., 1998; Thomson et al., 1998).

Microscopically, the disease is characterized by a mild to moderate catarrhal colitis with erosions of the surface epithelium and spirochaetal colonization. The presence of a fringe, 4-7 µm thick, of densely packed spirochaetes attached by one end perpendicular to the surface epithelium is a common feature of spontaneous infection and is recognized as intestinal spirochaetosis (Girard et al., 1995; Jacques et al., 1989; Taylor & Trott, 1997; Trott et al., 1996b). Evident disease with diarrhoea and colitis has been reproduced in experimental studies using different isolates of B. pilosicoli; however, intestinal spirochaetosis has hitherto only been revealed focally in a small number of the challenged pigs (Jensen et al., 2000; Taylor et al., 1980; Thomson et al., 1997; Trott et al., 1996b).

Six 6-week-old pigs weighing 12-14 kg (crossbreeds of Danish Landrace and Yorkshire), were obtained from the Danish Veterinary Institute's high health herd known to be free from Brachyspira hyodysenteriae and B. pilosicoli (Jensen et al., 2000). The animals were housed and fed as described previously by Jensen et al. (2000). Upon arrival at the experimental facilities, faeces samples from all animals were examined for pig-pathogenic Escherichia coli, haemolytic spirochaetes and Salmonella enterica (Møller et al., 1998). Inclusion of non-inoculated control pigs was intended, but we were unable to include the animals due to subclinical infection with S. enterica subsp. enterica serovar Typhimurium (S. Typhimurium) in the originating herd and a subsequent ban on transportation of the animals to the experimental facilities. A Danish isolate of B. pilosicoli (designated strain BP98-V), originally cultured from a pig with diarrhoea, was used for inoculation. The spirochaete was cultured as described by Jensen et al. (2000) and approximately 1x10⁹ c.f.u. was given to each pig by stomach tube daily for 3 consecutive days. The animals were monitored for diarrhoea, and rectally collected faecal samples were taken daily until the end of the trial at day 17 post-inoculation (p.i.).

Bacteriological examination of the faecal samples for B. pilosicoli and other pathogens (including S. enterica, B. hyodysenteriae and pathogenic E. coli) was performed as described previously by Møller et al. (1998) and Jensen et al. (2000). The animals were killed on successive days from day 8 p.i. Immediately after death, tissue samples for histology were taken from the small intestines, caecum, proximal colon and distal colon at the top of the spiral. The samples were fixed in 10 % buffered formalin and processed routinely. Sections of the large intestines were used for fluorescent in situ hybridization with the fluorescein isothiocyanate-labelled oligonucleotide probe Pilosi209 specific for B. pilosicoli (Boye et al., 1998); the presence of S. enterica was similarly investigated by in situ hybridization (Nordentoft et al., 1997).

A change in faecal consistency from normal to loose was already seen in the first pigs between day 1 and 3 p.i. (Table 1). The number of affected pigs and the severity of diarrhoea increased gradually. At day 8 p.i., while the other four pigs all had loose faeces, two had watery diarrhoea; these were sacrificed at day 8 p.i. Three of the four remaining pigs had persistent loose or watery diarrhoea until they were sacrificed at day 14 or 17 p.i.

Table 1. Faecal score, bacteriology and faecal re-isolation of B. pilosicoli from challenged pigs Faecal score: +++, watery; ++, semiliquid; +, semisolid. Bp, Isolation of B. pilosicoli; *, isolation of S. Typhimurium; †, animal sacrificed.

B. pilosicoli could be isolated from day 3 p.i. to the end of the study. Between day 5 and the end of the study at day 17 p.i., B. pilosicoli was isolated from 87 % (47/54) of the faecal samples. S. Typhimurium was isolated from 48 % (40/84) of the faecal samples between days 0 and 17 p.i., but only after pre-enrichment.

On post-mortem examination, the small intestines appeared normal, whereas the large intestines of all animals were slightly distended by gas and liquid contents. The mucosal surface appeared normal to mildly hyperaemic and without ulcerations. Small mucopurulent flecks were seen on the colonic surface of three pigs (Table 2). Although the gross lesions of the large intestines were subtle, diffuse catarrhal colitis with hyperplastic and elongated crypts and a mononuclear infiltrate into the lamina propria was revealed microscopically in all animals. A thin mucus layer mixed with desquamated enterocytes, mononuclear cells and neutrocytes could be seen covering parts of the mucosal surface. The mucosal surface itself showed sloughing of epithelial cells in the extrusion zone between the crypt openings, and multifocal erosions with exocytosis. In situ hybridization with Pilosi209 revealed a 4-7 µm thick fringe. This fringe, intestinal spirochaetosis, was most extensive in sections of the proximal and spiral colon, whereas it was observed in the caecum of only three animals. The fringe consisted of densely to moderate packed B. pilosicoli organisms, as illustrated in Fig. 1. Moreover, perpendicular end-attached single or loosely packed spirochaetes were similarly identified as B. pilosicoli by in situ hybridization (Fig. 2). In multiple foci with erosions, exocytosis and adherent ingesta, severe infection with spirochaetal invasion of the adjacent lamina propria could be found. In situ hybridization with the S. enterica-specific probe revealed no signals, and no lesions suggestive of salmonellosis were observed in any sections from the animals. Thus, although half of the faecal samples were Salmonella-positive by culture, we were convinced that the colitis in the challenged pigs was not associated with the presence of S. Typhimurium.

Table 2. Pathology and extent of intestinal spirochaetosis in B. pilosicoli-challenged pigs Intestinal spirochaetosis was defined by the presence of a fringe of end-on-attached B. pilosicoli and is scored as: +++, extensive; ++, multifocal; +, focal; -, not detected. NAD, No abnormalities detected; Col, catarrhal colitis.

(116K): Fig. 1. Colonic mucosa with extensive intestinal spirochaetosis. The fringe of densely packed spirochaetes was identified as B. pilosicoli organisms (green) by fluorescent in situ hybridization. Original magnification, x40; red/green filter set. The tissue appears coloured owing to autofluorescence; for instance, erythrocytes are orange spots.

(127K): Fig. 2. Demonstration by fluorescent in situ hybridization of focal intestinal spirochaetosis and solitary B. pilosicoli organisms (arrows) attached end-on to the colonic epithelium. Original magnification, x63; green filter.

The present study showed a relatively early onset of diarrhoea and, even more significantly, the development of extensive intestinal spirochaetosis in the colon of all animals compared with previous challenge studies, in which the end-on attachment of spirochaetes has been observed focally in only a few pigs (Jensen et al., 2000; Taylor et al., 1980; Thomson et al., 1997; Trott et al., 1996b). The end-on attachment of B. pilosicoli to the colonic epithelium in large numbers as well as observation of solitary end-attached spirochaetes suggests that the phenomenon of intestinal spirochaetosis is independent of the density of the organism on the colonic surface, but could involve specific mechanisms. So far, there is no information about the nature of the attachment mechanism or of possible receptors on the epithelial cell membrane (Taylor & Trott, 1997). The occurrence of end-attached spirochaetes has been associated with the early pathogenesis of B. pilosicoli infection (Taylor & Trott, 1997); however, the extensive intestinal spirochaetosis in this study was revealed in animals sacrificed 1-12 days after onset of diarrhoea. Compared with our previous study, in which only two of six pigs showed transient diarrhoea and one showed focal intestinal spirochaetosis (Jensen et al., 2000), we used another isolate in the present study and the animals, although originating from the same herd, were 2 weeks younger (6 instead of 8 weeks old). Both the age of the animals and the strain are features that could be important for the development of intestinal spirochaetosis. Even though the colonic mucosa microscopically was affected with severe spirochaetal infection, the gross alterations of the large intestines were subtle and could easily be not noticed or ignored. Thus, besides verifying the pathogenicity of B. pilosicoli, this study demonstrates the diagnostic importance of appropriate sampling in cases of suspicion of porcine spirochaetal colitis, whether or not gross lesions are present.