Open Access

Neither Hippurate-negative Brachyspira pilosicoli nor Brachyspira pilosicoli Type Strain Caused Diarrhoea in Early-weaned Pigs by Experimental Infection

  • M Fossi1,
  • K Ahlsten2,
  • T Pohjanvirta3,
  • M Anttila4,
  • T Kokkonen4,
  • TK Jensen5,
  • M Boye5,
  • A Sukura6,
  • K Pelkola7 and
  • S Pelkonen3
Acta Veterinaria Scandinavica200546:257

https://doi.org/10.1186/1751-0147-46-257

Received: 10 January 2005

Accepted: 01 September 2005

Published: 31 December 2005

Abstract

A hippurate-negative biovariant of Brachyspira pilosicoli (B. pilosicolihipp-) is occasionally isolated in diarrhoeic pigs in Finland, often concomitantly with hippurate-positive B. pilosicoli or Lawsonia intracellularis. We studied pathogenicity of B. pilosicolihipp- with special attention paid to avoiding co-infection with other enteric pathogens. Pigs were weaned and moved to barrier facilities at the age of 11 days. At 46 days, 8 pigs were inoculated with B. pilosicolihipp- strain Br1622, 8 pigs were inoculated with B. pilosicoli type strain P43/6/78 and 7 pigs were sham-inoculated. No signs of spirochaetal diarrhoea were detected; only one pig, inoculated with P43/6/78, had soft faeces from day 9 to 10 post inoculation. The pigs were necropsied between days 7 and 23 after inoculation. Live pigs were culture-negative for Brachyspira spp., but B. pilosicolihipp- was reisolated from necropsy samples of two pigs. The lesions on large colons were minor and did not significantly differ between the three trial groups. In silver-stained sections, invasive spirochaetes were detected in colonic mucosae of several pigs in all groups. Fluorescent in situ hybridisation for genus Brachyspira, B. pilosicoli and strain Br1622 was negative. However, in situ detection for members of the genus Leptospira was positive for spirochaete-like bacteria in the colonic epithelium of several pigs in both infected groups as well as in the control group. L. intracellularis, Salmonella spp., Yersinia spp. and intestinal parasites were not detected. The failure of B. pilosicoli strains to cause diarrhoea is discussed with respect to infectivity of the challenge strains, absence of certain intestinal pathogens and feed and management factors.

Keywords

Brachyspira pilosicoli intestinal spirochaetosis spirochaetal colitis pig diarrhoea experimental infection early weaning Lawsonia intracellularis

Sammanfattning

Varken hippurat negativa Brachyspira pilosicoli eller Brachyspira pilosicoli typ stammar framkallade diarré hos tidigt avvanda grisar vid experimentell infektion.

Den hippurat negativa biovarianten av Brachyspirapilosicoli (B. pilosicolihipp-) kan ofta isoleras ur diarrésjukagrisar i Finland, vanligen tillsammans medhippurat positiva B. pilosicoli eller Lawsonia intracellularis.Vi undersökte hur patogen B. pilosicolihippär,och tog notis om andra enteropatogener. Grisar avvandesoch avskiljdes vid 11 dygns ålder. Vid 46dygns ålder inokulerades 8 grisar med B. pilosicolihipp-, 8 grisar med B. pilosicoli stam P43/6/78 och på7 grisar utfördes inokuleringsproceduren utan bakterier(skeninokulation). Mellan 7 och 23 dygn efterinokuleringen avlivades och obducerades grisarna.Endast en (1) gris som blivit inokulerad med stammenP43/6/78 hade lindrig diarré från och med dygn9 till och med dygn 10 efter inokulering. De levandegrisarna var odlingsnegativa för B. pilosicoli, medanB. pilosicolihipp- kunde återisoleras i obduktionsmaterialetpå två grisar. Endast lindriga tarmförändringarobserverades och dessa skiljde sig inte signifikantfrån varandra mellan de tre prövningsgrupperna.I tjocktarmen från grisar i alla gruppersågs under mikroskop invasiva spiroketer. Tre olikaoligonukleotidprober användes för att identifieraBrachyspira med fluorescens in situ hybridisering,men dessa prov utföll negativt. I kolonepiteliet hoshälften av grisarna gav emellertid användningen aven prob för Leptospira positivt utslag. Avsaknaden avL. intracellularis påvisades ytterligare med serologiskatest och med artspecifik innesluten PCR(nested PCR). B. pilosicoli-stammar kunde såledesinte framkalla diarré och detta diskuteras i förhållandetill saminfektioner med vissa tarmpatogeneroch i förhållande till en eventuell enteropatogenlindrandeeffekt hos B. pilosicoli.

Introduction

Porcine intestinal spirochaetosis (PIS) in weaned pigs is caused by an anaerobic, weakly β-haemolytic spirochaete, Brachyspira pilosicoli [31, 35], and it occurs worldwide [7, 23, 3, 12, 6, 5]. The pathogenicity of different B. pilosicoli strains has been shown to vary [32]. B. pilosicoli usually hydrolyses hippurate, which is a major criterion for the biochemical differentiation of B. pilosicoli from the other porcine Brachyspira species [8]. However, some porcine B. pilosicoli strains in Finland are hippurate-negative and their pathogenicity is unclear since other potentially enteropathogenic bacteria, such as Lawsonia intracellularis and B. pilosicoli with a positive hippurate reaction, have simultaneously been diagnosed in the herds [10].

Co-infections with L. intracellularis and B. pilosicoli are highly prevalent among weaned pigs with diarrhoea in Sweden [13], and co-infections with L. intracellularis and various species of Brachyspira have also been reported in Denmark and the UK [23, 33]. The concomitant ileitis caused by L. intracellularis or colitis due to the pathogenic species of Brachyspira aggravates the clinical outcome. L. intracellularis and pathogenic species of Brachyspira characteristically cause diarrhoea in pigs after weaning. The time-point of the intestinal colonisation by these organisms during the nursing period is unknown.

In this work, we evaluated the pathogenicity of a single strain of hippurate-negative B. pilosicoli (B. pilosicolihipp-), paying particular attention to the simultaneous presence or absence of other enteric pathogens. The pigs were weaned and moved to barrier facilities at the age of 11 days to enable the effect of early separation on transmission of opportunistic pathogens from sows to piglets to be assessed.

Materials and methods

The experimental infection was approved by the Ethics Committee for Animal Experiments of the National Veterinary and Food Research Institute and carried out under license number 2-2002. Piglets were purchased from a herd of 120 sows with a long history of good health status. Freedom from swine enzootic pneumonia, atrophic rhinitis and Salmonella was verified by regular laboratory investigations. Several studies for Brachyspira spp. performed within the last two years had occasionally yielded weakly β-haemolytic spirochaetes other than B. pilosicoli.

On the farm, nine sows were moved to a farrowing department two weeks before the expected farrowing date. Colostrum samples were collected on the day of farrowing, and faecal samples were taken two days after farrowing. Twelve Landrace piglets and 12 mixed-breed piglets were chosen from four litters which had been born within 24 hours of each other. Equal numbers of male and female piglets for the both races were chosen. No creep feed was served before weaning. At the age of 11 days, the piglets were transported to barrier facilities of the Finnish National Veterinary and Food Research Institute.

The piglets were raised together in a pen of 6.20 m2 until the age of 45 days. The pen had a solid floor which was scraped clean daily. The peat bedding was changed to wood shavings 4–7 days post-inoculation (p.i.). The piglets were fed a commercial sow milk substitute until the age of 24 days. Commercial creep feed was served from the age of 13 to 31 days. Commercial grower feed was based on wheat and barley until the age of 57 days, and thereafter, on barley. The crude protein content of the initial feed was 17.2%, and from the age of 57 days onwards, 17.4%. No growth-promoting feed additives were used, and the feed was granulated without heating.

Most piglets had diarrhoea at the age of 15–18 days. Abundant growth of haemolytic Escherichia coli was observed in faecal cultures during this period. One piglet died. All piglets were medicated intramuscularly with trime to-prime-sulphadiazine at the age of 16 and 17 days. At 45 days of age, the pigs were divided into three trial groups in which littermates, sex and race were evenly distributed. Two groups of eight pigs were placed in two identical isolated rooms, where pen space per pig was 0.60 m2. The third group of seven pigs was divided into two subgroups of four and three pigs and placed in two smaller rooms with pen spaces per pig of 0.68 m2 and 0.51 m2.

The health of the pigs was monitored daily. Body temperature and weight gain were recorded, and faecal samples were initially taken weekly, and after the challenge, three times a week. Blood samples were collected weekly.

Challenge

B. pilosicolihipp- strain Br1622 had originally been isolated in the year 2000 from a pig herd in which weaners and young fatteners had diarrhoea. B. pilosicoli type strain P43/6/78 (ATCC 51139T) originated from a diarrhoeic pig in the UK [31]. The strains were stored at -70°C in beef broth supplemented with 12% horse serum and 15% glycerol.

Br1622 and P43/6/78 were cultured on fastidious anaerobe agar and incubated anaerobically for three days at 39°C. The next passages were propagated anaerobically at 39°C in brain heart infusion broth supplemented with 10% foetal calf serum and 0.5% glucose. Broth cultures were propagated through three passages for a final volume of 300 ml. The final broth was incubated for 36 ± 3 hours to achieve a density of 1.6–2.0 × 108 cells ml-1. The second cascade of broth cultures was started one day after the first one.

At the ages of 46 and 47 days, the eight pigs in one room were inoculated intragastrically with 3.2 × 109 cells of Br1622 on both days, and the eight pigs in the second room received 4.0 × 109 and 3.2 × 109 cells of P43/6/78 on two consecutive days. The seven pigs in the two small rooms were sham-inoculated with a sterile broth on both days. Bacterial viability in the broth containers was controlled after inoculation by microscopy and cultivation.

Necropsy

One pig from both challenge groups and the control group was euthanised and necropsied as blind on days 7, 9, 11, 15, 16, 17, 21 and 23 p.i. Blood samples were taken and the intestines were dissected immediately after the bolt stunning. The routine gross examination was performed paying special attention to intestinal mucosae. The caecum, the ascending, mid-spiral and descending colon, the rectum and the intestinal lymph nodes were sampled for histopathology.

The tissue samples were fixed in 10% buffered formalin, processed routinely, sectioned at 5 μm and stained with haematoxylin and eosin, and Warthin-Starry silver stain. The sections were blind-examined. For transmission electron microscopy (TEM), 1.0 mm2 samples from the mucosa of the large intestine were fixed in 2.5% glutaraldehyde in phosphate buffer. The samples were embedded in Epon™, and thin sections were stained with uranyl acetate and lead citrate. Samples were then visualised using a Jeol Jem 100-s electron microscope.

Mucosal scrapings for Brachyspira cultivation were taken from the caecum, ascending, mid-spiral and descending colon and rectum. Content from the large intestine and ileum was collected for further microbiological examination.

Fluorescent in situ hybridisation

The sections from the large colon were studied by fluorescent in situ hybridisation (FISH). The oligonucleotide probes used are presented in Table 2. The general bacterial probe EUB338, the genus-specific probe for Brachyspira and the species-specific probe for B. pilosicoli have been described elsewhere [4]. The strain-specific probe for Br1622, the specific probe for Treponema (pallidum group) and the genus-specific probes for Leptospira and Borrelia were selected using ARB software [30]. The probes were 5'-labelled with fluorescein isothiocyanate (MWG-Biotech AG, Ebersberg, Germany). The probe for strain Br1622 had at least two mismatches to all other Brachyspira species in the ARB database. Processing of the sections and hybridisation were carried out as described previously [16]. An Axioplan2 epifluorescence microscope was used for simultaneous detection of red and green fluorescence.
Table 2

Names, sequences and specificities of rRNA-targeted oligonucleotide probes used.

Probe

Systematic name1

Sequence (5'-3')

Specificity

Eub3382

S-D-Bact-0338-a-A-18

GCTGCCTCCCGTAGGAGT

Domain bacteria

NON-Eub3382

S-*-non-0338-a-S-18

CGACGGAGGGCATCCTCA

Unspecific control probe

SER14103

L-G-Brachy-1410-a-A-19

GTCATTCCATCGAAACATA

Genus Brachyspira

Pilosi2093

S-S-B.pilo-0209-a-A-18

GCTCATCGTGAAGCGAAA

B. pilosicoli

Br1622

S-S-Br1622-0637-a-A-18

CCAAGATCTACAGTATCC

Br1622

Treponema

S-G-Trepon-0728-a-A-18

TCGGCCAGAAACCCGCCT

Treponema spp.

Borrelia

S-G-Borre-0688-a-A-18

TATCAACAGATTCCACCC

Borrelia spp.

Leptospira

S-G-Leptospi-1414-a-A-18

CGGGTGCTCCCCACTCAG

Leptospira spp.

1 According to the Oligonucleotide Probe Database (OPD) nomenclature [1].

2 [2].

3 [3].

Microbiology, parasitology and haematology

The selective culture and the biochemical differentiation for Brachyspira spp. were performed as previously described [14, 8, 26]. The primary cultures from intestinal scrapings were studied also by B. pilosicoli-specific polymerase chain reaction (PCR) assays targeting 16S rDNA and 23S rDNA [9, 20]. The PCR studies were repeated with frozen gut samples when invasive spirochaetes were observed by histopathology. Pulsed-field gel electrophoresis (PFGE) with MluI restriction enzyme was used to compare DNA macro-restriction profiles between isolated spirochaetes and the challenge strains, as described earlier [11]. The banding patterns of DNA were compared visually.

Faecal and intestinal samples were cultured for facultative anaerobic bacteria and anaerobic bacteria, selectively for Campylobacter spp. and by enrichment methods for Salmonella spp. and Yersinia spp., using standard laboratory methods. Cultivation for Campylobacter spp. was done only from colon and caecum scrapings at necropsy.

Faecal samples of the sows and 26-day-old piglets were tested for L. intracellularis by nested PCR. Sample preparation was performed according to [22], and primer pairs used for DNA amplification were as described by [17]. Antibodies for L. intracellularis were analysed by using an indirect fluorescent antibody test (IFAT) [21, 19, 18] from the sera of pigs at the age of 42 days and at necropsy. L. intracellularis antibodies were analysed also from the colostrum of the pigs' dams, as well as from the colostrum of the other five sows farrowing in the same department during the same time.

Faecal samples from the challenge groups were pooled and investigated for rotavirus by Ani™ Rotatest (Ani Biotech Ltd., Vantaa, Finland) according to the manufacturer's instructions, and for parasite eggs by the flotation method. Blood samples were studied for total and differential leucocyte count, haematocrit value, haemoglobin concentration and platelet count.

Results

After the challenge, the pigs remained clinically healthy. Only one pig (pig no. 13, Table 1) inoculated with B. pilosicoli P43/6/78, had soft faeces on days 9 and 10 p.i. Intestinal spirochaetes were not isolated from this pig. During the first week p.i. the mean daily weight gain (DWG) was 0.48, 0.39 and 0.49 kg in the Br1622-, P43/6/78- and sham-inoculated groups, respectively. During the second week, the corresponding mean DWG was 0.58, 0.44 and 0.55 kg. The difference in mean DWG between the groups was statistically not significant. The body temperatures of the pigs remained within the normal range throughout the trial period.
Table 1

Pigs inoculated with B. pilosicolihipp- strain Br1622, B. pilosicoli type strain P43/6/78 or sterile broth (control). Pathology and results from fluorescent in situ hybridisation are shown.

Pig No.

Inoculum

Necropsy day post-inoculation

Gross pathology1

Histology

FISH4

    

Histopathology2

Spiral-shaped bacteria3

1

2

1

Br1622

7

-

+

-

+

-

2

Br1622

9

-

(+)

+

-

-

3

Br1622

11

+

+

++

+

-

4

Br1622

15

++

+

++, s

+

-

5

Br1622

16

++

(+)

-, s

+

-

6

Br1622

17

+

+

++

+

-

7

Br1622

21

+

(+)

++

-

-

8

Br1622

23

++

+

-

-

-

9

P43/6/78

7

+

(+)

+

-

-

10

P43/6/78

9

-

(+)

-

+

-

11

P43/6/78

11

+

(+)

+

-

-

12

P43/6/78

15

++

(+)

++

-

-

13

P43/6/78

16

++

(+)

++

+

-

14

P43/6/78

17

+

+

+

-

-

15

P43/6/78

21

+

(+)

++

+

-

16

P43/6/78

23

++

(+)

-

-

-

17

control

7

-

(+)

-

-

-

18

control

15

+

+

-

+

-

19

control

17

-

(+)

++

+

-

20

control

23

+

+

-

-

-

21

control

9

+

(+)

-

-

-

22

control

11

-

(+)

-

-

-

23

control

21

+

(+)

++

+

-

1Gross findings in caecum and/or colon. - = normal; + = mucosa slightly hyperaemic; ++ = mucosa hyperaemic and slightly oedematous.

2(+) = diffuse lymphocytic and plasmacytic infiltration in lamina propria, and multifocal accumulation of phagocytic macrophages beneath the epithelial cells of the caecum and/or colon; + = in addition, occasional microabscesses in crypts and/or mild exocytosis of neutrophils.

3Spiral-shaped bacteria in caecum and/or colon in silver-stained sections. - = none; + = very rare or irregularly observed in crypts and/or near tips of villi; ++ = invading through epithelial lining into lamina propria and/or abundantly in crypts; s = Br1622 reisolated from necropsy samples.

4Fluorescent in situ hybridisation. Positive (+) and negative (-) results with probes designed for 1 = domain bacteria and genus Leptospira, and 2 = genus Brachyspira, species B. pilosicoli and strain Br1622.

Pathology

At necropsy, a moderate hyperaemia was noted on colonic and/or caecal mucosae of six, seven and four pigs infected with Br1622, P43/6/78 and sterile broth, respectively. The colonic mucosae were also slightly oedematous in three pigs infected with Br1622 as well as in three pigs infected with P43/6/78 (Table 1). In light microscopy, minor inflammatory lesions were seen in sections of the large colon of all pigs (Table 1), but the severity of the microscopic lesions did not differ notably between the groups. In silver-stained sections from the large intestine, spiral-shaped bacteria were seen in the epithelium of five, six and two pigs in the Br1622, P43/6/78 and control groups, respectively. Spiral-shaped bacteria were more common in sections from the colon than from the caecum. The spiral-shaped bacteria crossed the epithelial cell layer into the lamina propria of the caecum and/or colon in samples from four, three and two pigs in these groups, respectively (Figs 1 and 2). Invasion of spiral-shaped bacteria below the lamina muscularis mucosae of the colon was seen in pig no.13, which was inoculated with P43/6/78. In TEM, trans-sections of end-oflagellated bacteria were seen between and below the colonic epithelial cells of one pig inoculated with Br1622 (no. 6) and one control pig (no. 23). The bacteria were 0.28–0.32 μm in diameter and had 10–14 endoflagella.
Figure 1

Invasion of spirochaetes in and below the colonic epithelium is shown in pig no. 13, which was inoculated with B. pilosicoli P43/6/78 and necropsied 16 days post-inoculation. Warthin-Starry silver staining. 1000×.

Figure 2

Invasive spirochaetes in a colonic crypt. Same pig as in Fig. 1. Warthin-Starry silver staining. 1000×.

Fluorescent in situ hybridisation

FISH with probes specific for Br1622, B. pilosicoli and the genus Brachyspira did not give a signal in any section of the large intestines. However, hybridisation with the probes for genus Leptospira and for domain bacteria revealed multiple spiral-shaped bacteria in and below the epithelial lining of the colon and/or caecum in five, three and three pigs in the groups inoculated with Br1622, P43/6/78 and sterile broth, respectively (Fig. 3). These spiral-shaped bacteria seemed uniform and were 6–11 μm in length.
Figure 3

Fluorescent in situ hybridisation with a genus-specific probe Leptospira. Demonstration of spirochaetes infiltrating the colonic epithelium. Pig no. 3 inoculated with B. pilosicolihipp- strain Br1622 and necropsied 11 days post-inoculation. 1000×.

Microbiology, parasitology and haematology

Brachyspira spp. were not isolated from the faecal samples of live pigs, but B. pilosicolihipp- was recovered from colon scrapings of two pigs challenged with Br1622 (pig nos. 4 and 5, Table 1) and necropsied on day 15 or 16 p.i. According to the PFGE macrorestriction profiles, the B. pilosicolihipp- isolates were the same as those of the challenge strain Br1622. By B. pilosicoli-specific PCR, all primary cultures for Brachyspira, excluding these two Brachyspira isolations, were negative.

Haemolytic E. coli was isolated from the faeces of most pigs until one week before challenge. Neither Salmonella spp. nor Yersinia spp. were isolated. Campylobacter coli or Campylobacter spp. were isolated from colon scrapings of four, five and four pigs inoculated with Br1622, P43/6/78 and sterile broth, respectively. Intestinal parasites were not detected. The latex test for rotavirus was positive in the group infected with Br1622 from the age of 46 days onwards, and in all other groups from the age of 48 days onwards. Beyond the age of 55 days, the test results became ambiguous.

Pigs' sera were negative for L. intracellularis antibodies. Four of the nine sows in the same farrowing group had colostral antibodies to L. intracellularis. However, these sows were not the dams of the trial pigs. L. intracellularis was not detected by PCR in faeces of the sows or the pigs.

The leucocyte count of one pig in both inoculated groups and two pigs in the control group exceeded the upper limit of reference (20 000 cells μl-1) once in the first week p.i. The values of the other haematological parameters remained within the normal range throughout the trial period.

Discussion

Neither B. pilosicoli type strain nor hippurate-negative field strain Br1622 caused diarrhoea in pigs. Reisolation of Br1622 succeeded for only two pigs at necropsy, whereas type strain P43/6/78 was not detected in any of the samples. The inoculated pigs possibly may have shed spirochaetes below the detection limits. The sensitivity of the cultivation for B. pilosicoli is approximated as 5.4 × 105 CFU g-1 faeces (Stege et al. 2000) or 1.5 × 102 CFU g-1 faeces [9]. The sensitivity of our cultivation method lies between these figures (unpublished data). In our experience, PCR from a primary culture rarely yields a positive result in cases where the culture has been negative for Brachyspira.

One interpretation is that true colonisation did not occur in most of the pigs. An oral inoculation of 109 cells ml-1 of B. pilosicoli given once [32, 34], twice [24] or thrice [15] has been satisfactory for induction of spirochaetal diarrhoea. Thus, the bacteria doses here should have been sufficient. However, undefined factors may exist that affect the actively growing bacteria inoculated as a pure suspension in an empty stomach.

An experimental infection by B. pilosicoli type strain P43/6/78 has been described to cause diarrhoea in gnotobiotic pigs [24]. This strain isolated and reported by [31], was later deposited in the American Type Culture Collection as a type strain of species B. pilosicoli [35]. An attenuation of this strain before its deposition can not be excluded. Strain Br1622 has undergone from four to six passages before its deposition, and a further four passages for the final volume of inocula. We assume that strain Br1622 is no more attenuated than the type strain at the stage of inoculation in this experiment. Some loss of pathogenicity of Br1622 through the passages can not, however, be excluded.

No infection of pigs by L. intracellularis was detected. Their early separation from the sows and the apparently low prevalence of infection in the herd of origin could explain the freedom from L. intracellularis. Co-infections, especially with L. intracellularis and B. pilosicoli, have been suggested to aggravate clinical diarrhoea among growers in field conditions [33, 13]. The absence of L. intracellularis could also promote the health of the pigs during the trial. Several environmental and management-related risk factors which enhance the outcome of diarrhoea among weaners were missing in this trial. The feed was not heat-treated and its crude protein content was relatively low. Heat treatment of feed during pelleting increases the prevalence of weakly β-haemolytic intestinal spirochaetes [29] and non-specific colitis in herds [28]. Moreover, high protein content in feed has been shown to cause loose stools in pigs [25]. The feed used in this trial might have supported the clinical health of the pigs to some extent.

Peat bedding is popular due to its high capacity for fluid absorption and low pH. In this trial, the peat was changed to wood shavings at 4–7 days p.i. However, we can speculate that sour peat may have controlled the number of circulating spirochaetes at the early stage of infection. The other environmental conditions in the barrier facilities were undoubtedly more favourable than those on conventional farms, possibly promoting the health of the trial pigs. The ventilation in the barrier facilities was filtrated, and diurnal variation in room temperature and draught was minimal. The pen area per pig increased gradually after seven days p.i. due to the necropsy schedule, which might have decreased the number of circulating bacteria. Further experiments for pathogenicity of B. pilosicolihipp- strains should be done for several B. pilosicolihipp- strains of various genotypes in a conventional-like environment using an unattenuated porcine B. pilosicoli strain as a positive control.

The bacteria observed in TEM sections of two pigs were deemed not to belong to the species B. pilosicoli because of their inconsistent endoflagella count [27]. In FISH, the positive signal merely by the genus-specific probe Leptospira was somewhat unexpected. Further investigations are needed to clarify the taxonomic position of these intestinal bacteria.

Notes

Declarations

Acknowledgements

We thank senior researcher Kirsi Partanen of MTT Agrifood Research Finland for the animals and feed. Päivi Olonen, Anssi Isoniemi, Minttu Mikkonen, Raili Heinonen and Pirkko Niemelä are thanked for technical assistance.

This study was supported by the Finnish Ministry for Agriculture and Forestry and the Finnish Association for Food Animal Practitioners.

Authors’ Affiliations

(1)
National Veterinary and Food Research Institute, Seinäjoki Unit
(2)
National Veterinary and Food Research Institute, Department of Virology and Epidemiology
(3)
National Veterinary and Food Research Institute, Kuopio Department
(4)
National Veterinary and Food Research Institute, Department of Pathology
(5)
Danish Institute for Food and Veterinary Research
(6)
Department of Basic Veterinary Sciences, University of Helsinki
(7)
National Veterinary and Food Research Institute, Department of Bacteriology

References

  1. Alm EW, Oerther DW, Larsen N, Stahl DA, Raskin L: The oligonucleotide probe database. Appl Environ Microbiol. 1996, 62: 3557-3559.PubMed CentralPubMedGoogle Scholar
  2. Amann RI, Binder BJ, Olson RJ, Chrisholm SW, Devereux R, Stahl DA: Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol. 1990, 56: 1919-1925.PubMed CentralPubMedGoogle Scholar
  3. Barcellos DESN, Mathiesen MR, de Uzeda M, Kader IITA, Duhamel GE: Prevalence of Brachyspira species isolated from diarrhoeic pigs in Brazil. Vet Rec. 2000, 146: 398-403.View ArticlePubMedGoogle Scholar
  4. Boye M, Jensen TK, Møller K, Leser TD, Jorsal SE: Specific detection of the genus Serpulina, S. hyodysenteriae and S. pilosicoli in porcine intestines by fluorescent rDNA in situ hybridization. Mol Cell Probe. 1998, 12: 323-330. 10.1006/mcpr.1998.0193.View ArticleGoogle Scholar
  5. Choi C, Han DU, Kim J, Cho WS, Chung H-K, Jung T, Yoon BS, Chae C: Prevalence of Brachyspira pilosicoli in Korean pigs determined using a nested PCR. Vet Rec. 2002, 150: 217-218.View ArticlePubMedGoogle Scholar
  6. de Arriba ML, Duhamel GE, Vidal AB, Carvajal A, Pozo J, Rubio P: Porcine Colonic Spirochetosis in Spanish pig herds. Proceedings of the 17th IPVS Congress, Ames, Iowa, USA, June 2-5, 2002, Vol. 2, p. 373.Google Scholar
  7. Duhamel GE: Colonic spirochetosis caused by Serpulina pilosicoli. Large Animal Practice 1998, 19, 14-22.Google Scholar
  8. Fellström C, Gunnarsson A: Phenotypical characterization of intestinal spirochaetes isolated from swine. Res Vet Sci. 1995, 59: 1-4. 10.1016/0034-5288(95)90021-7.View ArticlePubMedGoogle Scholar
  9. Fellström C, Pettersson B, Thomson J, Gunnarsson A, Persson M, Johanssen KE: Identification of Serpulina species associated with porcine colitis by biochemical analysis and PCR. J Clin Microbiol. 1997, 35: 462-467.PubMed CentralPubMedGoogle Scholar
  10. Fossi M, Pohjanvirta T, Sukura A, Heinikainen S, Lindecrona R, Pelkonen S: Molecular and ultrastructural characterization of porcine hippurate-negative Brachyspira pilosicoli. J. Clin. Microbiol. 2004, 42, 3153-3158.Google Scholar
  11. Fossi M, Pohjanvirta T, Pelkonen S: Molecular epidemiological study of Brachyspira pilosicoli in Finnish sow herds. Epidemiol Infect. 2003, 131: 967-973. 10.1017/S0950268803008963.View ArticleGoogle Scholar
  12. Heinonen M, Fossi M, Jalli J-P, Saloniemi H, Tuovinen V: Detectability and prevalence of Brachyspira species in herds rearing health class feeder pigs in Finland. Vet Rec. 2000, 146: 343-347.View ArticlePubMedGoogle Scholar
  13. Jacobson M, Hård af Segerstadt C, Gunnarsson A, Fellström C, de Verdier Klingenberg K, Wallgren P, Jensen-Waern M: Diarrhoea in the growing pig – a comparison of clinical, morphological and microbial findings between animals from good and poor performing herds. Res Vet Sci. 2003, 74: 163-169. 10.1016/S0034-5288(02)00187-X.View ArticlePubMedGoogle Scholar
  14. Jenkinson SR, Wingar CR: Selective medium for the isolation of Treponema hyodysenteriae. Vet. Rec. 1981, 109, 384-385.Google Scholar
  15. Jensen TK, Boye M, Møller K: Extensive intestinal spirochaetosis in pigs challenged with Brachyspira pilosicoli. J. Med. Microbiol. 2004, 53, 309-312.Google Scholar
  16. Jensen TK, Møller K, Boye M, Leser TD, Jorsal SE: Scanning electron microscopy and fluorescent in situ hybridization of experimental Brachyspira (Serpulina) pilosicoli infection in growing pigs. Vet Pathol. 2000, 37: 22-32. 10.1354/vp.37-1-22.View ArticlePubMedGoogle Scholar
  17. Jones GF, Ward GE, Murtaugh MP, Lin G, Gebhart CJ: Enhanced detection of the intracellular organism of swine proliferative enteritis, Ileal symbiont intracellularis, in feces by polymerase chain reaction. J Clin Microbiol. 1993, 31: 2611-2615.PubMed CentralPubMedGoogle Scholar
  18. Knittel JP, Jordan DM, Schwartz KJ, Janke BH, Roof MB, McOrist S, Harris DL: Evaluation of antemortem polymerase chain reaction and serologic methods for detection of Lawsonia intracellularis-exposed pigs. Am J Vet Res. 1998, 59: 722-726.PubMedGoogle Scholar
  19. Lawson GHK, McOrist S, Rowland AC, McCartney E, Roberts L: Serological diagnosis of the porcine proliferative enteropathies: implications for aetiology and epidemiology. Vet Rec. 1988, 122: 554-557.View ArticlePubMedGoogle Scholar
  20. Leser TD, Møller K, Jensen TK, Jorsal SE: Specific detection of Serpulina hyodysenteriae and potentially pathogenic weakly β-haemolytic porcine intestinal spirochetes by polymerase chain reaction targeting 23S rDNA. Mol Cell Probe. 1997, 11: 363-372. 10.1006/mcpr.1997.0129.View ArticleGoogle Scholar
  21. McOrist S, Boid R, Lawson GH, McConnell I: Monoclonal antibodies to intracellular campylobacter-like organisms of the porcine proliferative enteropathies. Vet Rec. 1987, 121: 421-422.View ArticlePubMedGoogle Scholar
  22. McOrist S, Gebhart CJ, Lawson GHK: Polymerase chain reaction for diagnosis of porcine proliferative enteropathy. Vet Microbiol. 1994, 41: 205-212. 10.1016/0378-1135(94)90101-5.View ArticlePubMedGoogle Scholar
  23. Møller K, Jensen TK, Jorsal SE, Leser TD, Carstensen B: Detection of Lawsonia intracellularis, Serpulina hyodysenteriae, weakly beta-haemolytic spirochaetes, Salmonella enterica, and haemolytic Escherichia coli from swine herds with and without diarrhoea among growing pigs. Vet Microbiol. 1998, 62: 59-72. 10.1016/S0378-1135(98)00199-0.View ArticlePubMedGoogle Scholar
  24. Neef NA, Lysons RJ, Trott DJ, Hampson DJ, Jones PW, Morgan JH: Pathogenity of porcine intestinal spirochetes in gnotobiotic pigs. Infect Immun. 1994, 62: 2395-2403.PubMed CentralPubMedGoogle Scholar
  25. Reese DE: Nutrient deficiencies and excesses. Diseases of Swine. Edited by: Straw BE, D'Allaire S, Mengeling WL, Taylor DJ. 1999, Iowa State University Press, Ames, Iowa 50014, USA, 743-755. 8Google Scholar
  26. Olson LD: Enhanced isolation of Serpulina hyodysenteriae by using sliced agar media. J Clin Microbiol. 1996, 34: 2937-2941.PubMed CentralPubMedGoogle Scholar
  27. Sellwood R, Bland AP: Ultrastructure of Intestinal Spirochaetes. Intestinal Spirochaetes in Domestic Animals and Humans. Edited by: Hampson DJ, Stanton TB. 1997, CAB International, Wallingford, OXON OX10 8DE, UK, 109-121.Google Scholar
  28. Smith WJ, Nelson EP: Grower scour/non-specific colitis. Vet Rec. 1987, 121: 334-View ArticlePubMedGoogle Scholar
  29. Stege H, Jensen TK, Møller K, Bækbo P, Jorsal S: Risk factors for intestinal pathogens in Danish finishing pig herds. Prev Vet Med. 2001, 50: 153-164. 10.1016/S0167-5877(01)00194-5.View ArticlePubMedGoogle Scholar
  30. Strunk O, Gross O, Reichel B, May M, Hermann S, Stuckmann N, et al: [Online] ARB: a software environment for sequence data. Department of Microbiology, Technische Universität München, Munich, Germany. 2000, [http://www.mikro.biologie.tu-muenchen.de]Google Scholar
  31. Thomson JR, Smith WJ, Murray BP, McOrist S: Pathogenity of three strains of Serpulina pilosicoli in pigs with a naturally acquired intestinal flora. Infect Immun. 1997, 65: 3693-3700.PubMed CentralPubMedGoogle Scholar
  32. Thomson JR, Smith WJ, Murray BP: Investigations into field cases of porcine colitis with particular reference to infection with Serpulina pilosicoli. Vet. Rec. 1998, 142, 235-239.Google Scholar
  33. Thomson JR, Smith WJ, Murray BP, Dick JE, Sumption KJ: Porcine enteric spirochete infections in the UK: surveillance data and preliminary investigation of atypical isolates. Anim Health Res Review. 2001, 2: 31-36.Google Scholar
  34. Thomson JR, Smith WJ, Murray BP, Dick JE, Sumption KJ: Porcine enteric spirochete infections in the UK: surveillance data and preliminary investigation of atypical isolates. Anim. Health Res. Review 2001, 2, 31-36.Google Scholar
  35. Trott DJ, Stanton TB, Jensen NS, Duhamel GE, Johnson JL, Hampson DJ: Serpulina pilosicoli sp. nov., the agent of porcine intestinal spirochetosis. Int J Syst Bacteriol. 1996, 46: 206-215.View ArticlePubMedGoogle Scholar

Copyright

© The Author(s) 2002