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Influences of diet and vaccination on colonisation of pigs by the intestinal spirochaete Brachyspira (Serpulina) pilosicoli
Veterinary Microbiology 73 (2000) 75±84
Short communication
In¯uences of diet and vaccination on colonisation of pigs by the intestinal spirochaete Brachyspira (Serpulina) pilosicoli D.J. Hampson*, I.D. Robertson, T. La, S.L. Oxberry, D.W. Pethick Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia Received 23 July 1999; received in revised form 26 October 1999; accepted 9 November 1999
Abstract The purpose of this study was to determine whether methods used to control swine dysentery (SD), caused by the intestinal spirochaete Brachyspira (Serpulina) hyodysenteriae, would also be effective in controlling porcine intestinal spirochaetosis (PIS) caused by the related spirochaete Brachyspira (Serpulina) pilosicoli. Weaner pigs in Groups I (n8) and II (n6) received a standard weaner pig diet based on wheat and lupins, whilst Group III (n6) received an experimental diet based on cooked white rice and animal protein. Pigs in Group II were vaccinated intramuscularly twice at a 3-week-interval with a formalinised bacterin made from B. pilosicoli porcine strain 95/ 1000 resuspended in Freund's incomplete adjuvant. Eleven days later pigs in all groups were infected orally with 1010 cells of strain 95/1000 on three successive days. One control pig in Group I developed acute diarrhoea, and at post-mortem had a severe erosive colitis with end-on attachment of spirochaetes to the colonic epithelium. All other pigs developed transient mild diarrhoea and had moderate patchy colitis at post-mortem 3 weeks later. B. pilosicoli was isolated from the faeces of all pigs, except for one fed rice, and was isolated from the mesenteric nodes of three pigs from Group I and from one vaccinated pig in Group II. Consumption of the rice-based diet, but not vaccination, delayed and signi®cantly (p<0.001) reduced the onset of faecal excretion of B. pilosicoli after experimental challenge. Vaccination induced a primary and secondary serological response to B. pilosicoli, as measured using sonicated whole cells of strain 95/1000 as an ELISA plate coating antigen. Antibody titres in the vaccinated pigs then declined, despite intestinal colonisation by B. pilosicoli. Both groups of unvaccinated animals also failed to develop a postinfection increase in circulating antibody titres. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Brachyspira (Serpulina) pilosicoli; Spirochaetosis; Vaccination; Feeding and nutrition *
Corresponding author. Tel.: 61-8-9360-2287; fax: 61-8-9310-4144. E-mail address: [email protected] (D.J. Hampson) 0378-1135/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 2 0 0 - X
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1. Introduction Brachyspira (Serpulina) pilosicoli is an anaerobic intestinal spirochaete which was recently named as the aetiological agent of porcine intestinal spirochaetosis (PIS) (Trott et al., 1996b; Ochiai et al., 1997). Infection of the large intestine results in mild patchy colitis, with intermittent diarrhoea (Taylor et al., 1980; Trott et al., 1996a; Thomson et al., 1997). In early cases of the disease dense mats of the spirochaetes may be found attached by one cell end to the caecal and colonic lumenal epithelium (Taylor et al., 1980; Trott et al., 1996a; Thomson et al., 1997). The spirochaete also infects other species, including dogs (Duhamel et al., 1998), birds (McLaren et al., 1997) and human beings (Trott et al., 1997a; Trivett-Moore et al., 1998). To date few attempts have been made to develop means to control infections with B. pilosicoli. Where PIS is a problem in piggeries, animals are routinely treated with antimicrobials, although the disease tends to recur following withdrawal of treatment (Thomson et al., 1998; Hampson and Trott, 1999). In contrast to B. pilosicoli, numerous different methods have been employed to control the closely related intestinal spirochaete Brachyspira (Serpulina) hyodysenteriae, the aetiologic agent of a severe mucohaemorrhagic colitis called swine dysentery (SD) (Hampson et al., 1997; Ochiai et al., 1997; Harris et al., 1999). For example, a number of different vaccines have been developed for SD, ranging from simple bacterins to recombinant proteins (Fernie et al., 1983; Gabe et al., 1995). Of these, only bacterins have been used with any success to control experimental SD, and the results have been very variable (Fernie et al., 1983; Hampson et al., 1993). Protection is at least in part lipooligosaccharide (LOS)-serotype speci®c (Joens et al., 1983), where protection against SD has been achieved by vaccination. Both vaccinated pigs and those which have recovered from natural infections develop circulating antibody titres to LOS and to outer membrane proteins of B. hyodysenteriae (Chat®eld et al., 1988; Wannemuehler et al., 1988). On the other hand, little is known about immunity to B. pilosicoli in pigs or other species, and there have been no reports on the use of vaccines to control intestinal spirochaetosis. Recently it has been shown that feeding pigs a diet consisting of cooked white rice and animal protein offers protection from experimental infection with B. hyodysenteriae (Siba et al., 1996). The basis of this protection is incompletely understood, but this observation does present a novel approach to the control of SD. Given the close relatedness of B. pilosicoli and B. hyodysenteriae, including their similar ecological niches in the porcine large intestine, this study was undertaken to determine whether either the use of a bacterin vaccine or a rice-based diet would be helpful in reducing colonisation and/or protecting pigs from the clinical effects of B. pilosicoli infections. 2. Materials and methods 2.1. Animals and diets Twenty healthy piglets from a herd known to be free of SD and Salmonella spp. were weaned at 20 days of age and then transported to an isolation animal house at Murdoch
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University. They were randomly allocated to three groups and housed in a single room in adjacent pens separated by open wire mesh partitions that allowed close contact between the animals in the various groups. Pigs in Groups I (n8) and II (n6) were fed an antimicrobial-free unpelleted weaner ration which was based on wheat and lupins (20% crude protein, 14.7 MJ/kg digestible energy). Pigs in Group III (n6) received a diet consisting of freshly-cooked long grain white rice supplemented with animal protein, which was balanced to meet the energy and protein speci®cations of the wheat diet. Both diets have been described previously (Siba et al., 1996). The diets and fresh water were offered ad libitum. Pigs were weighed at weekly intervals throughout the experimental period. Faecal swabs were taken on the day of arrival to con®rm that the pigs were not infected with intestinal spirochaetes. These were inoculated onto agar plates selective for intestinal spirochaetes (Jenkinson and Wingar, 1981), and incubated at 378C for 5 days in an atmosphere of 94% H2 and 6% CO2 2.2. Preparation of vaccine A bacterin was prepared from B. pilosicoli strain 95/1000, using the methods previously employed to prepare a B. hyodysenteriae bacterin (Hampson et al., 1993). Strain 95/1000 was from a Western Australian pig which had died with post-mortem signs of intestinal spirochaetosis, and has previously been used in our laboratory to experimentally infect weaner pigs (Trott et al., 1996a). The strain was recovered from frozen stocks held at the Reference Centre for Intestinal Spirochaetes, Murdoch University, and grown to mid-log phase in 350 ml of Kunkle's prereduced anaerobic Trypticase soy broth (Kunkle et al., 1986). Cells were harvested by centrifugation, washed three times in sterile phosphate buffered saline pH 7.2 (PBS), then resuspended in PBS and inactivated by addition of 0.4% formalin followed by stirring overnight at 48C. The cells were then counted in a haemocytometer under a phase contrast microscope, adjusted to 1010 cells per ml, aliquoted and stored at ÿ208C. Immediately before administration the bacterin was thawed and emulsi®ed in an equal volume of Freund's Incomplete Adjuvant (CSL, Melbourne, Australia). The pigs in Group II were vaccinated with 2 ml of the preparation by deep intramuscular injection in the neck. They were vaccinated 3 days after weaning, and again 24 days later. 2.3. Sera Pigs were bled six times during the course of the experiment, commencing immediately prior to the ®rst immunisation, then on Days 21 (3 days prior to the second immunisation), 32 (on the day prior to infection), 40, 48 and 54 after the ®rst immunisation. The blood was held at 48C overnight then the serum was removed, aliquoted and stored at ÿ208C. Immediately prior to use the sera were thawed on ice and brie¯y vortexed. 2.4. Development of ELISA Whole cells of B. pilosicoli strain 95/1000 (108/ml in PBS) were sonicated on ice using three cycles of 30 s with 2 min between cycles. The sonicate was centrifuged at 10,000g
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for 10 min and the supernatant was used as plate coating antigen stock. Total protein content was determined using a commercial dye-binding assay kit (Bio-Rad Laboratories, Hercules, CA, USA), then the sonicate was diluted in bicarbonate/carbonate coating buffer (pH 9.6) to a working dilution of 1.6 mg total protein per ml. 100 ml of the working dilution was added to each well of a 96 well microtitre plate (Becton Dickinson, Lincoln Park, NJ, USA) and this was left at 48C overnight. The coating solution was tipped off and the plates were washed three times in PBS-0.05% (v/v) Tween 20 (PBST). Each serum sample was subjected to two-fold serial dilutions (1:20±1:1280) in PBST containing 1% (w/v) bovine serum albumin (Sigma, St Louis, MO, USA) and 100 ml added to each well. Plates were incubated at 258C for 2 h with gentle shaking, then washed three times with PBST. 100 ml of diluted (1:2000) goat anti-pig IgG horseradish peroxidase conjugate (Southern Biotechnology, Birmingham, AL, USA) was then added to each well and the plates incubated at 258C for 1 h with gentle shaking. The plates were washed three times with PBST and 100 ml of o-phenylenediamine dihydrochloride (OPD) (Sigma) substrate solution (0.1% w/v OPD, 0.03% H2O2, phosphate-citrate buffer, pH 5.0) was added to each well and incubated for 10 min at 258C. Colour development was stopped with 2 M H2SO4 before reading the optical density (OD) in an ELISA plate reader at 490 nm. 2.5. Experimental infection All pigs were challenged via stomach tube with approximately 1010 active viable cells of B. pilosicoli strain 95/1000 contained in 100 ml of Kunkle's broth, on each of three successive days starting on Day 33 after the ®rst immunisation (9 days after the second vaccination). Pigs were then monitored daily for signs of ill-health, particularly signs of diarrhoea, and faecal swabs were taken every 2±3 days and cultured on selective intestinal spirochaete medium, as previously described (Trott et al., 1996a). To con®rm their identity, four selected spirochaete isolates from infected pigs in each group were subjected to multilocus enzyme electrophoresis (MLEE), as previously described (Lee et al., 1993). 2.6. Post-mortem examination The pigs were euthanised in batches of two or three animals per day over a period starting 3 weeks after challenge. Pigs were euthanised by intravenous injection of 200 mg/kg body weight of sodium barbiturate (Valabarb, Jurox, Sydney, Australia). The abdominal cavity was opened and four to six mesenteric lymph nodes located immediately adjacent to the large intestine were excised and transferred to a sterile petri dish. Their surface was sprayed with 70% (v/v) alcohol, seared with a heated spatula, then each node was incised using a fresh sterile scalpel blade. A thin-necked cotton-tipped swab was inserted into the incisions and suf®cient lymph collected to saturate the cotton tip. This was then immediately plated to spirochaete selective agar. One node from each animal was placed into 10% (v/v) formalin for subsequent histological examination. The intestinal tract was then dissected out and the caecum and colon opened. Swabs were taken from the wall of the caecum and mid-colon and plated on selective agar for spirochaetes, and on MacConkey agar (Oxoid, Unipath, Australia).
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Fresh tissue from these two areas was also placed into 10% (v/v) formalin, and after ®xation and dehydration, histological sections (4 mm) were cut and stained with haematoxylin and eosin and with Steiner silver stain. 2.7. Statistical analysis Body weights were compared using Student's t-tests. Faecal excretion of B. pilosicoli and occurrence of diarrhoea were evaluated by Chi-square analysis. The number of sampling days where the organism was either isolated or not isolated, or diarrhoea was recorded or not recorded, were compared for the three groups. 3. Results 3.1. Clinical outcome One pig in Group I developed an acute severe watery diarrhoea 2 days after the third experimental inoculation. It was euthanised 2 days later because it was moribund. All the other pigs produced loose faeces on one or two sampling days. This did not occur at the same time after challenge in all piglets, nor were there any signi®cant differences in days of diarrhoea between the three groups. The mild diarrhoea generally coincided with periods of faecal shedding of B. pilosicoli. During the diarrhoeal period the animals did not show any other clinical symptoms. Group body weights were not signi®cantly different throughout the experiment. 3.2. Faecal shedding B. pilosicoli was isolated from the faeces of all but one rice-fed pig at some stage following experimental challenge. The duration of faecal shedding by individual pigs was quite variable, and in some cases it was also intermittent, with periods of up to 10 days between bouts of excretion. Precise daily ®gures were not available because samples were not taken every day, but overall the mean ®rst onset of faecal shedding in the pigs fed rice (Group III), expressed as days after the last day of experimental challenge, was 10 days (range 0±26), compared to 3 days (range 1±12) for the control pigs of Group I and 4 days (range 1±10) for the vaccinated pigs in Group II. The mean duration of faecal shedding for the rice fed pigs was 5 days (range 0±9) compared to 16 days (range 1±28) for the control pigs and 18.3 days (range 12±28) for the vaccinated animals. Days of faecal shedding for the control and vaccinated pigs were not signi®cantly different, but the pigs fed rice had signi®cantly fewer days of excretion than both the control pigs (w212.27; p<0.001) and the vaccinated pigs (w222.72; p<0.001). All the faecal isolates tested had the same electrophoretic type in MLEE as the inoculated B. pilosicoli strain 95/1000. 3.3. Post-mortem ®ndings The single pig with acute diarrhoea had severe multifocal ®brinonecrotic typhlocolitis, and the mesenteric nodes were enlarged. On histological examination there were severe
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Fig. 1. Histological section showing attachment of Brachyspira pilosicoli cells to degenerating colonic enterocytes in an experimentally infected pig with severe erosive ®brinonecrotic typhlocolitis. Bar1 mm.
erosions with complete denuding of the colonic epithelium and extensive in®ltration of lesions with polymorphic neutrophils and lymphoid cells. Large numbers of protozoa resembling Balantidium coli were distributed through the debris on the lumenal surface. The intact epithelium adjacent to the eroded areas was carpeted with a thick covering of B. pilosicoli cells attached by one cell end to the epithelium (Fig. 1). The single B. pilosicoli isolate tested from the lesion had the same electrophoretic pro®le as the inoculated strain. Post-mortem showed that the other pigs had gross evidence of a mild patchy colitis. Histological examination showed a moderately severe subacute mucosal colitis. Major features were an expansion of the lamina propria with lymphocytes and plasma cells, and sometimes oedema, a multifocal crypt neutrophilia, a high crypt mitotic rate, loss of or mucus extrusion from goblet cells, and focal attenuation of surface epithelium. The lymph nodes were generally reactive. There were no consistent differences in ®ndings between pigs in the three groups. Despite isolating B. pilosicoli from the caecum and colon of most animals at post-mortem examination, no end-on attachment was identi®ed in the histological sections taken from these sites. B. pilosicoli was isolated from the mesenteric nodes of three of the unvaccinated control pigs fed wheat (including the acutely affected animal), and from one vaccinated animal. 3.4. ELISA titres The serum titres were determined as the dilution at which the OD490 nm would be zero, as calculated from the linear regression of the log10(time) versus OD490 nm plot. Mean serum titres for the pigs in each group were plotted against time post-vaccination (Fig. 2). Vaccinated pigs showed a primary and secondary antibody response following the two vaccinations. Titres were highest at the time of bacterial challenge, however these subsequently declined despite the animals remaining colonised. Titres for the pigs in the other two groups remained low throughout the experimental period.
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Fig. 2. Mean serum antibody levels of vaccinated and unvaccinated pigs challenged with B. pilosicoli strain 95/ 1000. Vaccinated pigs (±*±) fed on a wheat-based diet were given two intramuscular injections at Days 0 and 24. Unvaccinated pigs were fed either a wheat-based diet (±~±) or a rice-based diet (±&±). All pigs were orally challenged on three successive days with B. pilosicoli strain 95/1000 between Days 33 and 35. Standard error bars are shown.
4. Discussion From an experimental point of view the model of PIS used here was successful. Pigs on a standard diet became colonised for an extended period following experimental challenge, and developed a mild transient diarrhoea. This is consistent with observations in the ®eld, and with the ®ndings of other workers who have infected pigs under experimental circumstances (Taylor et al., 1980; Trott et al., 1996a; Thomson et al., 1997). Interestingly, one of the six control pigs developed an acute watery diarrhoea with severe erosive colitis soon after experimental challenge. The presence of large numbers of spirochaetes adjacent to the lesions, and in some cases associated with degenerating enterocytes (Fig. 1), suggests that the organisms provoked the reaction. The presence of Balantidium coli as a secondary invader of colonic lesions in pigs is a common ®nding (Hampson et al., 1997; Hampson and Trott, 1999). The other pigs showed less severe changes, and no end-on attachment of spirochaetes was observed at post-mortem 3 weeks after challenge. In a previous study using strain 95/1000, 4 of 12 infected pigs developed obvious colitis. One pig had spirochaetes attached end-on to the colonic epithelium, whilst the others had spirochaetes in the colonic crypts, in association with crypt abscessation (Trott et al., 1996a). These differences in ®ndings might be explained by the fact that the affected pigs in the previous study were euthanised within 11 days of infection, whereas the animals in this study were kept for 24 days or more. Recovery of B. pilosicoli from the mesenteric nodes of pigs has not been recorded before, and is interesting as the related spirochaete B. hyodysenteriae has been considered
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not to invade beyond the lamina propria (Hampson et al., 1997). This site was sampled because it has been shown that immunocompromised human patients may develop a spirochaetemia with B. pilosicoli (Trott et al., 1997b), and it was presumed that the spirochaetes would need to pass through the regional nodes to reach the circulation. Recently, a related spirochaete: Brachyspira (Serpulina) murdochii has been isolated from the joint ¯uid of a pig with arthritis (Hampson et al., 1999), suggesting the possibility that intestinal spirochaetes in general may be a previously unrecognised cause of acute or chronic lesions at sites other than the intestine. The organisms are easy to overlook as they stain poorly with Gram stain, and they require special methods for isolation. Consuming a cooked rice-based diet signi®cantly retarded the ®rst appearance of B. pilosicoli in the faeces compared to pigs on a standard diet, but, unlike the situation with B. hyodysenteriae (Siba et al., 1996), intestinal colonisation was not completely prevented by the rice diet. Once colonisation of these pigs started it persisted to the end of the experiment, in the same way as seen in the pigs on the standard diet. Experimental studies with gnotobiotic piglets have shown that B. hyodysenteriae requires interaction with other anaerobic members of the colonic micro¯ora before it can effectively colonise (Mayer et al., 1975; Whipp et al., 1979). The rice-based diet is thought to protect against SD because it provides little substrate for the micro¯ora in the large intestine, which in turn then is unable to support the growth of B. hyodysenteriae (Pluske et al., 1996; Durmic et al., 1998). The current results suggest that, despite occupying similar niches in the large intestine, the two pathogenic spirochaete species have different biological properties, with B. hyodysenteriae having more specialised growth requirements in the colon than B. pilosicoli. Nevertheless, the rice-based diet did signi®cantly delay B. pilosicoli establishment and shedding. Besides altering fermentation patterns in the large intestine, the rice-based diet also results in physical changes such as sparser and drier large intestinal contents, and it could be that these effects contributed to the dif®culty and delay in establishing colonisation. The fact that colonisation eventually occurred in ®ve of the six pigs may also have been facilitated by repeated exposure to faeces from the infected pigs in the other two groups, which were housed in adjacent pens. Although the duration of shedding by pigs fed the rice-based diet was shorter than that of the pigs on the other two diets, in part this may have been because the experiment was terminated before faecal shedding by all pigs had ceased. Preparation and use of an autogenous bacterin was based on procedures for B. hyodysenteriae strains, which have been used with variable success to help control SD in experimentally infected pigs (Fernie et al., 1983; Hampson et al., 1993). The bacterin induced primary and secondary antibody responses as measured by ELISA, but did not reduce intestinal colonisation, occurrence of diarrhoea or colonic changes compared to unvaccinated pigs. Only one vaccinated pig had B. pilosicoli recovered from its mesenteric nodes, compared to three of the control pigs, but the numbers of animals used were too low to be reliable as an indicator of whether or not the vaccine had prevented dissemination or reduced spirochaete viability after translocation of the organisms from the intestinal tract. It is not altogether surprising that a systemic vaccine had no effect in terms of providing protection in the colon, even though such preparations give a degree of protection against SD. In SD there is a marked erosive colitis in most animals, and it is
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likely that systemic antibody will reach and may provide some degree of protection at the site of the main lesion. At this stage little is known about the antigenic composition of the outer surface of B. pilosicoli, other than that it has an antigenic LOS which is smoother than that of B. hyodysenteriae (Lee and Hampson, 1999). Clearly more needs to be known about the antigenic characteristics of the organism and the local and systemic immune responses of infected animals to it before effective vaccines can be developed. An interesting observation was that antibody titres tended to decrease following vaccination, despite the presence of spirochaetes in the intestinal lumen. Similarly the unvaccinated animals in both the other groups did not develop signi®cant titres to B. pilosicoli, despite being colonised with the organism, and in some cases having the spirochaete in their mesenteric nodes. This apparent lack of a systemic immune response may create problems for the future development of diagnostic serological tests for B. pilosicoli. Acknowledgements This study was supported by a grant from the Australian Pig Research and Development Corporation. References Chat®eld, S.N., Fernie, D.S., Penn, C., Dougan, G., 1988. Identi®cation of the major antigens of Treponema hyodysenteriae and comparison with those of Treponema innocens. Infect. Immun. 56, 1070±1075. Duhamel, G.E., Trott, D.J., Muniappa, N., Mathiesen, M.R., Tarasiuk, K., Lee, J.I., Hampson, D.J., 1998. Canine intestinal spirochetes consist of Serpulina pilosicoli and a newly identi®ed group provisionally designated Serpulina canis. J. Clin. Microbiol. 36, 2264±2270. Durmic, Z., Pethick, D.W., Pluske, J.R., Hampson, D.J., 1998. Changes in bacterial populations in the colon of pigs fed different sources of dietary ®bre, and the development of swine dysentery after experimental challenge. J. Appl. Microbiol. 85, 574±582. Fernie, D.S., Ripley, P.H., Walker, P.D., 1983. Swine dysentery: protection against experimental challenge following single dose parenteral immunisation with inactivated Treponema hyodysenteriae. Res. Vet. Sci. 35, 217±221. Gabe, J.D., Chang, R.J., Slomiany, R., Andrews, W.H., McCaman, M.T., 1995. Isolation of extracytoplasmic proteins from Serpulina hyodysenteriae B204 and molecular cloning of the ¯aB1 gene encoding a 38kilodalton ¯agellar protein. Infect. Immun. 63, 142±148. Hampson, D.J., Trott, D.J., 1999. Spirochetal diarrhea/porcine intestinal spirochetosis. In: Straw, B.E., D'Allaire, S., Mengeling, W.L., Taylor , D.J. (Eds.), Diseases of Swine, 8th Edition. Blackwell Scienti®c Publications, Oxford, U.K. pp. 553±562. Hampson, D.J., Robertson, I.D., Mhoma, J.R.L., 1993. Experiences with a vaccine being developed for the control of swine dysentery. Aust. Vet. J. 70, 18±20. Hampson, D.J., Atyeo, R.F., Combs, B.G., 1997. Swine dysentery. In: Hampson, D.J., Stanton, T.B. (Eds.), Intestinal Spirochaetes in Domestic Animals and Humans. CAB International, Wallingford, U.K. pp. 175±209. Hampson, D.J., Robertson, I.D., Oxberry, S.L., 1999. Isolation of Serpulina murdochii from the joint ¯uid of a lame pig. Aust. Vet. J. 77, 48. Harris, D.L., Hampson, D.J., Glock, R.D., 1999. Swine dysentery. In: Straw, B.E., D'Allaire, S., Mengeling, W.L., Taylor , D.J. (Eds.), Diseases of Swine, 8th Edition. Blackwell Scienti®c Publications, Oxford, U.K. pp. 579±600.
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Jenkinson, S.R., Wingar, C.R., 1981. Selective medium for the isolation of Treponema hyodysenteriae. Vet. Rec. 109, 384±385. Joens, L.A., Whipp, S.C., Glock, R.D., Nuessen, M.E., 1983. Serotype-speci®c protection against Treponema hyodysenteriae infection in ligated loops of pigs recovered from swine dysentery. Infect. Immun. 39, 460± 462. Kunkle, R.A., Harris, D.L., Kinyon, J.M., 1986. Autoclaved liquid medium for propagation of Treponema hyodysenteriae. J. Clin. Microbiol. 24, 669±671. Lee, J.I., Hampson, D.J., Lymbery, A.J., Harders, S.J., 1993. The porcine intestinal spirochaetes: identi®cation of new genetic groups. Vet. Microbiol. 34, 273±285. Lee, B.J., Hampson, D.J., 1999. Lipooligosaccharide pro®les of Serpulina pilosicoli strains, and their serological cross-reactivities. J. Med. Microbiol. 48, 411±415. McLaren, A.J., Trott, D.J., Swayne, D.E., Oxberry, S.L., Hampson, D.J., 1997. Genetic and phenotypic characterisation of intestinal spirochetes colonizing chickens, and allocation of known pathogenic isolates to three distinct genetic groups. J. Clin. Microbiol. 35, 412±417. Mayer, R.C., Simons, J., Byerly, C.S., 1975. The etiology of swine dysentery. III. The role of selected gram negative obligate anaerobes. Vet. Pathol. 12, 46±54. Ochiai, S., Mori, K., Adachi, Y., 1997. Uni®cation of the genera Serpulina and Brachyspira, and proposals of Brachyspira hyodysenteriae comb nov, Brachyspira innocens comb nov and Brachyspira pilosicoli comb nov. Microbiol. Immunol. 41, 445±452. Pluske, J.R., Siba, P.M., Pethick, D.W., Durmic, Z., Mullan, B.P., Hampson, D.J., 1996. The incidence of swine dysentery in pigs can be reduced by feeding diets that limit fermentation in the large intestine. J. Nutr. 126, 2920±2933. Siba, P.M., Pethick, D.W., Hampson, D.J., 1996. Pigs experimentally infected with Serpulina hyodysenteriae can be protected from developing swine dysentery by feeding them a highly digestible diet. Epidemiol. Infect. 116, 207±216. Taylor, D.J., Simmons, J.R., Laird, A.M., 1980. Production of diarrhoea and dysentery in pigs by feeding pure cultures of a spirochaete differing from Treponema hyodysenteriae. Vet. Rec. 106, 326±332. Thomson, J.R., Smith, W.J., Murray, B.P., McOrist, S., 1997. Pathogenicity of three strains of Serpulina pilosicoli in pigs with a naturally acquired intestinal ¯ora. Infect. Immun. 65, 3693±3700. Thomson, J.R., Smith, W.J., Murray, B.P., 1998. Investigations into ®eld cases of porcine colitis with particular reference to infection with Serpulina pilosicoli. Vet. Rec. 142, 235±239. Trott, D.J., Huxtable, C.R., Hampson, D.J., 1996a. Experimental infection of newly weaned pigs with human and porcine strains of Serpulina pilosicoli. Infect. Immun. 64, 4648±4654. Trott, D.J., Stanton, T.B., Jensen, N.S., Duhamel, G.E., Johnson, J., Hampson, D.J., 1996b. Serpulina pilosicoli sp. nov., the agent of porcine intestinal spirochetosis. Int. J. System. Bacteriol. 46, 206±215. Trott, D.J., Combs, B.G., Oxberry, S.L., Mikosza, A.S.J., Robertson, I.D., Passey, M., Taime, J., Sehuko, R., Hampson, D.J., 1997a. The prevalence of Serpulina pilosicoli in humans and domestic animals in the Eastern Highlands of Papua New Guinea. Epidemiol. Infect. 119, 369±379. Trott, D.J., Jensen, N.S., Saint Girons, I., Oxberry, S.L., Stanton, T.B., Lindquist, D., Hampson, D.J., 1997b. Identi®cation and characterisation of Serpulina pilosicoli isolates from the blood of critically-ill patients. J. Clin. Microbiol. 35, 482±485. Trivett-Moore, N.L., Gilbert, G.L., Law, C.L.H., Trott, D.J., Hampson, D.J., 1998. Isolation of Serpulina pilosicoli from rectal biopsy specimens showing evidence of intestinal spirochetosis. J. Clin. Microbiol. 36, 261±265. Wannemuehler, M.J., Hubbard, R.D., Greer, J.M., 1988. Characterization of the major outer membrane antigens of Treponema hyodysenteriae. Infect. Immun. 56, 3032±3039. Whipp, S.C., Robinson, I.M., Harris, D.L., Glock, R.D., Mathews, P.J., Alexander, T.J.L., 1979. Pathogenic synergism between Treponema hyodysenteriae and other selected anaerobes in gnotobiotic pigs. Infect. Immun. 26, 1042±1047.
Short communication
In¯uences of diet and vaccination on colonisation of pigs by the intestinal spirochaete Brachyspira (Serpulina) pilosicoli D.J. Hampson*, I.D. Robertson, T. La, S.L. Oxberry, D.W. Pethick Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia Received 23 July 1999; received in revised form 26 October 1999; accepted 9 November 1999
Abstract The purpose of this study was to determine whether methods used to control swine dysentery (SD), caused by the intestinal spirochaete Brachyspira (Serpulina) hyodysenteriae, would also be effective in controlling porcine intestinal spirochaetosis (PIS) caused by the related spirochaete Brachyspira (Serpulina) pilosicoli. Weaner pigs in Groups I (n8) and II (n6) received a standard weaner pig diet based on wheat and lupins, whilst Group III (n6) received an experimental diet based on cooked white rice and animal protein. Pigs in Group II were vaccinated intramuscularly twice at a 3-week-interval with a formalinised bacterin made from B. pilosicoli porcine strain 95/ 1000 resuspended in Freund's incomplete adjuvant. Eleven days later pigs in all groups were infected orally with 1010 cells of strain 95/1000 on three successive days. One control pig in Group I developed acute diarrhoea, and at post-mortem had a severe erosive colitis with end-on attachment of spirochaetes to the colonic epithelium. All other pigs developed transient mild diarrhoea and had moderate patchy colitis at post-mortem 3 weeks later. B. pilosicoli was isolated from the faeces of all pigs, except for one fed rice, and was isolated from the mesenteric nodes of three pigs from Group I and from one vaccinated pig in Group II. Consumption of the rice-based diet, but not vaccination, delayed and signi®cantly (p<0.001) reduced the onset of faecal excretion of B. pilosicoli after experimental challenge. Vaccination induced a primary and secondary serological response to B. pilosicoli, as measured using sonicated whole cells of strain 95/1000 as an ELISA plate coating antigen. Antibody titres in the vaccinated pigs then declined, despite intestinal colonisation by B. pilosicoli. Both groups of unvaccinated animals also failed to develop a postinfection increase in circulating antibody titres. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Brachyspira (Serpulina) pilosicoli; Spirochaetosis; Vaccination; Feeding and nutrition *
Corresponding author. Tel.: 61-8-9360-2287; fax: 61-8-9310-4144. E-mail address: [email protected] (D.J. Hampson) 0378-1135/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 2 0 0 - X
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1. Introduction Brachyspira (Serpulina) pilosicoli is an anaerobic intestinal spirochaete which was recently named as the aetiological agent of porcine intestinal spirochaetosis (PIS) (Trott et al., 1996b; Ochiai et al., 1997). Infection of the large intestine results in mild patchy colitis, with intermittent diarrhoea (Taylor et al., 1980; Trott et al., 1996a; Thomson et al., 1997). In early cases of the disease dense mats of the spirochaetes may be found attached by one cell end to the caecal and colonic lumenal epithelium (Taylor et al., 1980; Trott et al., 1996a; Thomson et al., 1997). The spirochaete also infects other species, including dogs (Duhamel et al., 1998), birds (McLaren et al., 1997) and human beings (Trott et al., 1997a; Trivett-Moore et al., 1998). To date few attempts have been made to develop means to control infections with B. pilosicoli. Where PIS is a problem in piggeries, animals are routinely treated with antimicrobials, although the disease tends to recur following withdrawal of treatment (Thomson et al., 1998; Hampson and Trott, 1999). In contrast to B. pilosicoli, numerous different methods have been employed to control the closely related intestinal spirochaete Brachyspira (Serpulina) hyodysenteriae, the aetiologic agent of a severe mucohaemorrhagic colitis called swine dysentery (SD) (Hampson et al., 1997; Ochiai et al., 1997; Harris et al., 1999). For example, a number of different vaccines have been developed for SD, ranging from simple bacterins to recombinant proteins (Fernie et al., 1983; Gabe et al., 1995). Of these, only bacterins have been used with any success to control experimental SD, and the results have been very variable (Fernie et al., 1983; Hampson et al., 1993). Protection is at least in part lipooligosaccharide (LOS)-serotype speci®c (Joens et al., 1983), where protection against SD has been achieved by vaccination. Both vaccinated pigs and those which have recovered from natural infections develop circulating antibody titres to LOS and to outer membrane proteins of B. hyodysenteriae (Chat®eld et al., 1988; Wannemuehler et al., 1988). On the other hand, little is known about immunity to B. pilosicoli in pigs or other species, and there have been no reports on the use of vaccines to control intestinal spirochaetosis. Recently it has been shown that feeding pigs a diet consisting of cooked white rice and animal protein offers protection from experimental infection with B. hyodysenteriae (Siba et al., 1996). The basis of this protection is incompletely understood, but this observation does present a novel approach to the control of SD. Given the close relatedness of B. pilosicoli and B. hyodysenteriae, including their similar ecological niches in the porcine large intestine, this study was undertaken to determine whether either the use of a bacterin vaccine or a rice-based diet would be helpful in reducing colonisation and/or protecting pigs from the clinical effects of B. pilosicoli infections. 2. Materials and methods 2.1. Animals and diets Twenty healthy piglets from a herd known to be free of SD and Salmonella spp. were weaned at 20 days of age and then transported to an isolation animal house at Murdoch
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University. They were randomly allocated to three groups and housed in a single room in adjacent pens separated by open wire mesh partitions that allowed close contact between the animals in the various groups. Pigs in Groups I (n8) and II (n6) were fed an antimicrobial-free unpelleted weaner ration which was based on wheat and lupins (20% crude protein, 14.7 MJ/kg digestible energy). Pigs in Group III (n6) received a diet consisting of freshly-cooked long grain white rice supplemented with animal protein, which was balanced to meet the energy and protein speci®cations of the wheat diet. Both diets have been described previously (Siba et al., 1996). The diets and fresh water were offered ad libitum. Pigs were weighed at weekly intervals throughout the experimental period. Faecal swabs were taken on the day of arrival to con®rm that the pigs were not infected with intestinal spirochaetes. These were inoculated onto agar plates selective for intestinal spirochaetes (Jenkinson and Wingar, 1981), and incubated at 378C for 5 days in an atmosphere of 94% H2 and 6% CO2 2.2. Preparation of vaccine A bacterin was prepared from B. pilosicoli strain 95/1000, using the methods previously employed to prepare a B. hyodysenteriae bacterin (Hampson et al., 1993). Strain 95/1000 was from a Western Australian pig which had died with post-mortem signs of intestinal spirochaetosis, and has previously been used in our laboratory to experimentally infect weaner pigs (Trott et al., 1996a). The strain was recovered from frozen stocks held at the Reference Centre for Intestinal Spirochaetes, Murdoch University, and grown to mid-log phase in 350 ml of Kunkle's prereduced anaerobic Trypticase soy broth (Kunkle et al., 1986). Cells were harvested by centrifugation, washed three times in sterile phosphate buffered saline pH 7.2 (PBS), then resuspended in PBS and inactivated by addition of 0.4% formalin followed by stirring overnight at 48C. The cells were then counted in a haemocytometer under a phase contrast microscope, adjusted to 1010 cells per ml, aliquoted and stored at ÿ208C. Immediately before administration the bacterin was thawed and emulsi®ed in an equal volume of Freund's Incomplete Adjuvant (CSL, Melbourne, Australia). The pigs in Group II were vaccinated with 2 ml of the preparation by deep intramuscular injection in the neck. They were vaccinated 3 days after weaning, and again 24 days later. 2.3. Sera Pigs were bled six times during the course of the experiment, commencing immediately prior to the ®rst immunisation, then on Days 21 (3 days prior to the second immunisation), 32 (on the day prior to infection), 40, 48 and 54 after the ®rst immunisation. The blood was held at 48C overnight then the serum was removed, aliquoted and stored at ÿ208C. Immediately prior to use the sera were thawed on ice and brie¯y vortexed. 2.4. Development of ELISA Whole cells of B. pilosicoli strain 95/1000 (108/ml in PBS) were sonicated on ice using three cycles of 30 s with 2 min between cycles. The sonicate was centrifuged at 10,000g
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for 10 min and the supernatant was used as plate coating antigen stock. Total protein content was determined using a commercial dye-binding assay kit (Bio-Rad Laboratories, Hercules, CA, USA), then the sonicate was diluted in bicarbonate/carbonate coating buffer (pH 9.6) to a working dilution of 1.6 mg total protein per ml. 100 ml of the working dilution was added to each well of a 96 well microtitre plate (Becton Dickinson, Lincoln Park, NJ, USA) and this was left at 48C overnight. The coating solution was tipped off and the plates were washed three times in PBS-0.05% (v/v) Tween 20 (PBST). Each serum sample was subjected to two-fold serial dilutions (1:20±1:1280) in PBST containing 1% (w/v) bovine serum albumin (Sigma, St Louis, MO, USA) and 100 ml added to each well. Plates were incubated at 258C for 2 h with gentle shaking, then washed three times with PBST. 100 ml of diluted (1:2000) goat anti-pig IgG horseradish peroxidase conjugate (Southern Biotechnology, Birmingham, AL, USA) was then added to each well and the plates incubated at 258C for 1 h with gentle shaking. The plates were washed three times with PBST and 100 ml of o-phenylenediamine dihydrochloride (OPD) (Sigma) substrate solution (0.1% w/v OPD, 0.03% H2O2, phosphate-citrate buffer, pH 5.0) was added to each well and incubated for 10 min at 258C. Colour development was stopped with 2 M H2SO4 before reading the optical density (OD) in an ELISA plate reader at 490 nm. 2.5. Experimental infection All pigs were challenged via stomach tube with approximately 1010 active viable cells of B. pilosicoli strain 95/1000 contained in 100 ml of Kunkle's broth, on each of three successive days starting on Day 33 after the ®rst immunisation (9 days after the second vaccination). Pigs were then monitored daily for signs of ill-health, particularly signs of diarrhoea, and faecal swabs were taken every 2±3 days and cultured on selective intestinal spirochaete medium, as previously described (Trott et al., 1996a). To con®rm their identity, four selected spirochaete isolates from infected pigs in each group were subjected to multilocus enzyme electrophoresis (MLEE), as previously described (Lee et al., 1993). 2.6. Post-mortem examination The pigs were euthanised in batches of two or three animals per day over a period starting 3 weeks after challenge. Pigs were euthanised by intravenous injection of 200 mg/kg body weight of sodium barbiturate (Valabarb, Jurox, Sydney, Australia). The abdominal cavity was opened and four to six mesenteric lymph nodes located immediately adjacent to the large intestine were excised and transferred to a sterile petri dish. Their surface was sprayed with 70% (v/v) alcohol, seared with a heated spatula, then each node was incised using a fresh sterile scalpel blade. A thin-necked cotton-tipped swab was inserted into the incisions and suf®cient lymph collected to saturate the cotton tip. This was then immediately plated to spirochaete selective agar. One node from each animal was placed into 10% (v/v) formalin for subsequent histological examination. The intestinal tract was then dissected out and the caecum and colon opened. Swabs were taken from the wall of the caecum and mid-colon and plated on selective agar for spirochaetes, and on MacConkey agar (Oxoid, Unipath, Australia).
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Fresh tissue from these two areas was also placed into 10% (v/v) formalin, and after ®xation and dehydration, histological sections (4 mm) were cut and stained with haematoxylin and eosin and with Steiner silver stain. 2.7. Statistical analysis Body weights were compared using Student's t-tests. Faecal excretion of B. pilosicoli and occurrence of diarrhoea were evaluated by Chi-square analysis. The number of sampling days where the organism was either isolated or not isolated, or diarrhoea was recorded or not recorded, were compared for the three groups. 3. Results 3.1. Clinical outcome One pig in Group I developed an acute severe watery diarrhoea 2 days after the third experimental inoculation. It was euthanised 2 days later because it was moribund. All the other pigs produced loose faeces on one or two sampling days. This did not occur at the same time after challenge in all piglets, nor were there any signi®cant differences in days of diarrhoea between the three groups. The mild diarrhoea generally coincided with periods of faecal shedding of B. pilosicoli. During the diarrhoeal period the animals did not show any other clinical symptoms. Group body weights were not signi®cantly different throughout the experiment. 3.2. Faecal shedding B. pilosicoli was isolated from the faeces of all but one rice-fed pig at some stage following experimental challenge. The duration of faecal shedding by individual pigs was quite variable, and in some cases it was also intermittent, with periods of up to 10 days between bouts of excretion. Precise daily ®gures were not available because samples were not taken every day, but overall the mean ®rst onset of faecal shedding in the pigs fed rice (Group III), expressed as days after the last day of experimental challenge, was 10 days (range 0±26), compared to 3 days (range 1±12) for the control pigs of Group I and 4 days (range 1±10) for the vaccinated pigs in Group II. The mean duration of faecal shedding for the rice fed pigs was 5 days (range 0±9) compared to 16 days (range 1±28) for the control pigs and 18.3 days (range 12±28) for the vaccinated animals. Days of faecal shedding for the control and vaccinated pigs were not signi®cantly different, but the pigs fed rice had signi®cantly fewer days of excretion than both the control pigs (w212.27; p<0.001) and the vaccinated pigs (w222.72; p<0.001). All the faecal isolates tested had the same electrophoretic type in MLEE as the inoculated B. pilosicoli strain 95/1000. 3.3. Post-mortem ®ndings The single pig with acute diarrhoea had severe multifocal ®brinonecrotic typhlocolitis, and the mesenteric nodes were enlarged. On histological examination there were severe
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Fig. 1. Histological section showing attachment of Brachyspira pilosicoli cells to degenerating colonic enterocytes in an experimentally infected pig with severe erosive ®brinonecrotic typhlocolitis. Bar1 mm.
erosions with complete denuding of the colonic epithelium and extensive in®ltration of lesions with polymorphic neutrophils and lymphoid cells. Large numbers of protozoa resembling Balantidium coli were distributed through the debris on the lumenal surface. The intact epithelium adjacent to the eroded areas was carpeted with a thick covering of B. pilosicoli cells attached by one cell end to the epithelium (Fig. 1). The single B. pilosicoli isolate tested from the lesion had the same electrophoretic pro®le as the inoculated strain. Post-mortem showed that the other pigs had gross evidence of a mild patchy colitis. Histological examination showed a moderately severe subacute mucosal colitis. Major features were an expansion of the lamina propria with lymphocytes and plasma cells, and sometimes oedema, a multifocal crypt neutrophilia, a high crypt mitotic rate, loss of or mucus extrusion from goblet cells, and focal attenuation of surface epithelium. The lymph nodes were generally reactive. There were no consistent differences in ®ndings between pigs in the three groups. Despite isolating B. pilosicoli from the caecum and colon of most animals at post-mortem examination, no end-on attachment was identi®ed in the histological sections taken from these sites. B. pilosicoli was isolated from the mesenteric nodes of three of the unvaccinated control pigs fed wheat (including the acutely affected animal), and from one vaccinated animal. 3.4. ELISA titres The serum titres were determined as the dilution at which the OD490 nm would be zero, as calculated from the linear regression of the log10(time) versus OD490 nm plot. Mean serum titres for the pigs in each group were plotted against time post-vaccination (Fig. 2). Vaccinated pigs showed a primary and secondary antibody response following the two vaccinations. Titres were highest at the time of bacterial challenge, however these subsequently declined despite the animals remaining colonised. Titres for the pigs in the other two groups remained low throughout the experimental period.
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Fig. 2. Mean serum antibody levels of vaccinated and unvaccinated pigs challenged with B. pilosicoli strain 95/ 1000. Vaccinated pigs (±*±) fed on a wheat-based diet were given two intramuscular injections at Days 0 and 24. Unvaccinated pigs were fed either a wheat-based diet (±~±) or a rice-based diet (±&±). All pigs were orally challenged on three successive days with B. pilosicoli strain 95/1000 between Days 33 and 35. Standard error bars are shown.
4. Discussion From an experimental point of view the model of PIS used here was successful. Pigs on a standard diet became colonised for an extended period following experimental challenge, and developed a mild transient diarrhoea. This is consistent with observations in the ®eld, and with the ®ndings of other workers who have infected pigs under experimental circumstances (Taylor et al., 1980; Trott et al., 1996a; Thomson et al., 1997). Interestingly, one of the six control pigs developed an acute watery diarrhoea with severe erosive colitis soon after experimental challenge. The presence of large numbers of spirochaetes adjacent to the lesions, and in some cases associated with degenerating enterocytes (Fig. 1), suggests that the organisms provoked the reaction. The presence of Balantidium coli as a secondary invader of colonic lesions in pigs is a common ®nding (Hampson et al., 1997; Hampson and Trott, 1999). The other pigs showed less severe changes, and no end-on attachment of spirochaetes was observed at post-mortem 3 weeks after challenge. In a previous study using strain 95/1000, 4 of 12 infected pigs developed obvious colitis. One pig had spirochaetes attached end-on to the colonic epithelium, whilst the others had spirochaetes in the colonic crypts, in association with crypt abscessation (Trott et al., 1996a). These differences in ®ndings might be explained by the fact that the affected pigs in the previous study were euthanised within 11 days of infection, whereas the animals in this study were kept for 24 days or more. Recovery of B. pilosicoli from the mesenteric nodes of pigs has not been recorded before, and is interesting as the related spirochaete B. hyodysenteriae has been considered
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not to invade beyond the lamina propria (Hampson et al., 1997). This site was sampled because it has been shown that immunocompromised human patients may develop a spirochaetemia with B. pilosicoli (Trott et al., 1997b), and it was presumed that the spirochaetes would need to pass through the regional nodes to reach the circulation. Recently, a related spirochaete: Brachyspira (Serpulina) murdochii has been isolated from the joint ¯uid of a pig with arthritis (Hampson et al., 1999), suggesting the possibility that intestinal spirochaetes in general may be a previously unrecognised cause of acute or chronic lesions at sites other than the intestine. The organisms are easy to overlook as they stain poorly with Gram stain, and they require special methods for isolation. Consuming a cooked rice-based diet signi®cantly retarded the ®rst appearance of B. pilosicoli in the faeces compared to pigs on a standard diet, but, unlike the situation with B. hyodysenteriae (Siba et al., 1996), intestinal colonisation was not completely prevented by the rice diet. Once colonisation of these pigs started it persisted to the end of the experiment, in the same way as seen in the pigs on the standard diet. Experimental studies with gnotobiotic piglets have shown that B. hyodysenteriae requires interaction with other anaerobic members of the colonic micro¯ora before it can effectively colonise (Mayer et al., 1975; Whipp et al., 1979). The rice-based diet is thought to protect against SD because it provides little substrate for the micro¯ora in the large intestine, which in turn then is unable to support the growth of B. hyodysenteriae (Pluske et al., 1996; Durmic et al., 1998). The current results suggest that, despite occupying similar niches in the large intestine, the two pathogenic spirochaete species have different biological properties, with B. hyodysenteriae having more specialised growth requirements in the colon than B. pilosicoli. Nevertheless, the rice-based diet did signi®cantly delay B. pilosicoli establishment and shedding. Besides altering fermentation patterns in the large intestine, the rice-based diet also results in physical changes such as sparser and drier large intestinal contents, and it could be that these effects contributed to the dif®culty and delay in establishing colonisation. The fact that colonisation eventually occurred in ®ve of the six pigs may also have been facilitated by repeated exposure to faeces from the infected pigs in the other two groups, which were housed in adjacent pens. Although the duration of shedding by pigs fed the rice-based diet was shorter than that of the pigs on the other two diets, in part this may have been because the experiment was terminated before faecal shedding by all pigs had ceased. Preparation and use of an autogenous bacterin was based on procedures for B. hyodysenteriae strains, which have been used with variable success to help control SD in experimentally infected pigs (Fernie et al., 1983; Hampson et al., 1993). The bacterin induced primary and secondary antibody responses as measured by ELISA, but did not reduce intestinal colonisation, occurrence of diarrhoea or colonic changes compared to unvaccinated pigs. Only one vaccinated pig had B. pilosicoli recovered from its mesenteric nodes, compared to three of the control pigs, but the numbers of animals used were too low to be reliable as an indicator of whether or not the vaccine had prevented dissemination or reduced spirochaete viability after translocation of the organisms from the intestinal tract. It is not altogether surprising that a systemic vaccine had no effect in terms of providing protection in the colon, even though such preparations give a degree of protection against SD. In SD there is a marked erosive colitis in most animals, and it is
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likely that systemic antibody will reach and may provide some degree of protection at the site of the main lesion. At this stage little is known about the antigenic composition of the outer surface of B. pilosicoli, other than that it has an antigenic LOS which is smoother than that of B. hyodysenteriae (Lee and Hampson, 1999). Clearly more needs to be known about the antigenic characteristics of the organism and the local and systemic immune responses of infected animals to it before effective vaccines can be developed. An interesting observation was that antibody titres tended to decrease following vaccination, despite the presence of spirochaetes in the intestinal lumen. Similarly the unvaccinated animals in both the other groups did not develop signi®cant titres to B. pilosicoli, despite being colonised with the organism, and in some cases having the spirochaete in their mesenteric nodes. This apparent lack of a systemic immune response may create problems for the future development of diagnostic serological tests for B. pilosicoli. Acknowledgements This study was supported by a grant from the Australian Pig Research and Development Corporation. References Chat®eld, S.N., Fernie, D.S., Penn, C., Dougan, G., 1988. Identi®cation of the major antigens of Treponema hyodysenteriae and comparison with those of Treponema innocens. Infect. Immun. 56, 1070±1075. Duhamel, G.E., Trott, D.J., Muniappa, N., Mathiesen, M.R., Tarasiuk, K., Lee, J.I., Hampson, D.J., 1998. Canine intestinal spirochetes consist of Serpulina pilosicoli and a newly identi®ed group provisionally designated Serpulina canis. J. Clin. Microbiol. 36, 2264±2270. Durmic, Z., Pethick, D.W., Pluske, J.R., Hampson, D.J., 1998. Changes in bacterial populations in the colon of pigs fed different sources of dietary ®bre, and the development of swine dysentery after experimental challenge. J. Appl. Microbiol. 85, 574±582. Fernie, D.S., Ripley, P.H., Walker, P.D., 1983. Swine dysentery: protection against experimental challenge following single dose parenteral immunisation with inactivated Treponema hyodysenteriae. Res. Vet. Sci. 35, 217±221. Gabe, J.D., Chang, R.J., Slomiany, R., Andrews, W.H., McCaman, M.T., 1995. Isolation of extracytoplasmic proteins from Serpulina hyodysenteriae B204 and molecular cloning of the ¯aB1 gene encoding a 38kilodalton ¯agellar protein. Infect. Immun. 63, 142±148. Hampson, D.J., Trott, D.J., 1999. Spirochetal diarrhea/porcine intestinal spirochetosis. In: Straw, B.E., D'Allaire, S., Mengeling, W.L., Taylor , D.J. (Eds.), Diseases of Swine, 8th Edition. Blackwell Scienti®c Publications, Oxford, U.K. pp. 553±562. Hampson, D.J., Robertson, I.D., Mhoma, J.R.L., 1993. Experiences with a vaccine being developed for the control of swine dysentery. Aust. Vet. J. 70, 18±20. Hampson, D.J., Atyeo, R.F., Combs, B.G., 1997. Swine dysentery. In: Hampson, D.J., Stanton, T.B. (Eds.), Intestinal Spirochaetes in Domestic Animals and Humans. CAB International, Wallingford, U.K. pp. 175±209. Hampson, D.J., Robertson, I.D., Oxberry, S.L., 1999. Isolation of Serpulina murdochii from the joint ¯uid of a lame pig. Aust. Vet. J. 77, 48. Harris, D.L., Hampson, D.J., Glock, R.D., 1999. Swine dysentery. In: Straw, B.E., D'Allaire, S., Mengeling, W.L., Taylor , D.J. (Eds.), Diseases of Swine, 8th Edition. Blackwell Scienti®c Publications, Oxford, U.K. pp. 579±600.
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