Download Epidemiological aspects and improved differential - E

National Veterinary and Food Research Institute (EELA)
Seinäjoki Regional Unit
and
University of Helsinki
Faculty of Veterinary Medicine
Department of Basic Veterinary Sciences
Division of Microbiology and Epidemiology
Epidemiological aspects
and improved differential diagnostics
of porcine Brachyspira pilosicoli
Marja Fossi
ACADEMIC DISSERTATION
To be presented
with the permission of the Faculty of Veterinary Medicine,
University of Helsinki,
for public examination in Auditorium Walter,
Agnes Sjöberginkatu 2, Helsinki,
on April 28th 2006, at 12 noon.
Supervised by:
Sinikka Pelkonen, DVM, PhD, Professor
National Veterinary and Food Research Institute (EELA)
Kuopio Department, Kuopio, Finland
Tuula Honkanen-Buzalski, DVM, PhD, Professor
National Veterinary and Food Research Institute (EELA)
Helsinki, Finland
Supervising Professor: Airi Palva, MSc, PhD, Professor
Department of Basic Veterinary Sciences
Division of Microbiology and Epidemiology
Faculty of Veterinary Medicine, University of Helsinki, Finland
Reviewed by:
Claes Fellström, DVM, PhD, Professor
Department of Medicine and Surgery
Faculty of Veterinary Medicine and Animal Science
Swedish University of Agricultural Sciences, Uppsala, Sweden
Jill Thomson, DVM, PhD, DipECVP, CertPM, MRCVS
Veterinary Centre Manager
Scottish Agricultural College, Veterinary Services, Edinburgh, UK
Opponent:
Marja-Liisa Hänninen, DVM, PhD, Professor
Department of Food and Environmental Hygiene
Faculty of Veterinary Medicine, University of Helsinki, Finland
ISSN 1458-6878
ISBN 952-5568-27-X (Print)
ISBN 952-5568-28-8 (PDF)
Yliopistopaino
Helsinki 2006
Contents
Acknowledgements
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List of original publications .
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Abbreviations
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2 Review of the literature .
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1 Introduction
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2.1
Taxonomy and general characteristics of intestinal spirochaetes
2.2
Characteristics of Brachyspira spp.
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Species in genus Brachyspira
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2.4
Brachyspira pilosicoli in pigs
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2.4.1
Specific features of Brachyspira pilosicoli
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2.4.2
Porcine intestinal spirochaetosis
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2.4.3
Detection of Brachyspira pilosicoli .
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2.4.3.1
Isolation methods
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2.4.3.2
Phenotypic differentiation
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2.4.3.3
Molecular identification
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2.4.3.4
Fingerprinting
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Transmission of infection
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Treatment and control of porcine intestinal spirochaetosis
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3 Aims of the study
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4 Materials and methods .
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4.1
Sampling (IV, V)
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4.2
Bacterial isolates and strains (I–V) .
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4.3
Isolation (IV, V) and phenotypic identification (I–V)
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4.4
Polymerase chain reaction (I–V)
4.5
Sequencing of 16S rDNA (III)
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4.6
Pulsed-field gel electrophoresis (I, II, IV, V) .
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4.7
Fluorescent in situ hybridization (IV)
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4.8
Transmission electron microscopy (II, IV)
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4.9
Antimicrobial susceptibility testing (V) .
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4.10 Study design for hippurate-negative B. pilosicoli infection trial (IV)
4.11 Actions taken for eradication of B. pilosicoli (V)
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5 Results
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5.1
Subspecies analysis of Brachyspira isolates by PFGE (I , II, IV, V)
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5.2
16S rDNA sequences and ultrastructure of hippurate-negative
Brachyspira pilosicoli (II)
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5.3
D-ribose utilization of porcine Brachyspira spp. (III)
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5.4
Experimental infection of pigs by B. pilosicoli hipp- strain Br1622
and type strain P43 (IV)
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Eradication of B. pilosicoli (V)
5.5
6 Discussion
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Subspecies analysis of Brachyspira isolates by PFGE
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Molecular epidemiology of B. pilosicoli by PFGE
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6.3
Characterization of hippurate-negative Brachyspira pilosicoli
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6.4
D-ribose test in differentiation of porcine Brachyspira spp.
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Aspects of experimental infection of pigs with B. pilosicoli strains
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Eradication of B. pilosicoli .
7 Conclusions
8 References
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Acknowledgements
These studies were mainly conducted at five locations of the National Veterinary and Food
Research Institute: Seinäjoki Unit, Kuopio Department, Department of Pathology, Department of Bacteriology and Department of Virology. Some parts of the project were performed at the Pathology Division of the Department of Basic Veterinary Sciences and at
the Saari Unit of the Faculty of Veterinary Medicine, University of Helsinki. This work was
financially supported by the Ministry of Agriculture and Forestry, the Foundation of Veterinary Science in Finland and the Finnish Association for Food Animal Practitioners.
I am deeply grateful to Professor Sinikka Pelkonen, Head of the Kuopio Department, for
her supervision and unceasing support throughout the Brachyspira studies. The General
Director, Professor Tuula Honkanen-Buzalski, is warmly thanked for guidance and wise
counsel, especially during the final preparation of the manuscript.
My sincere thanks are also extended to all of my co-authors:
– Tarja Pohjanvirta and Sirpa Heinikainen for their essential contributions to the molecular
studies and their patience,
– Professor Marjukka Anttila, Head of Department of Pathology, and Teija Kokkonen for
skilful assistance with pathology,
– Katja Ahlsten for sharing with me the difficult but interesting days around the infection
trial,
– Teresa Skrzypczak for fruitful collaboration in the laboratory and pleasant companionship while attending conferences,
– Kirsti Pelkola for her careful microbiological studies during the infection trial,
– Professor Antti Sukura, Head of the Pathology Division, for helping me with electron
microscopy,
– Mari Heinonen and Olli Peltoniemi for their arrangements and actions for the eradication
of B. pilosicoli and for control samplings in a sow herd,
– Danish colleagues Tim Kåre Jensen, Mette Boye and Rikke Lindecrona for the molecular
studies carried out at the Danish Institute for Food and Veterinary Research.
All of my colleagues and the technicians at the facilities involved deserve my warmest
thanks for their positive attitudes and generous assistance with all manner of things.
Veterinary Centre Manager Jill Thomson and Professor Claes Fellström are sincerely thanked
for reviewing the thesis and providing valuable comments to improve it. I also thank Carol
Ann Pelli for editing the English language of this manuscript and most of the original articles.
5
Abstract
Brachyspira pilosicoli causes porcine intestinal spirochaetosis, which is manifested by a
mild, persistent diarrhoea among weaned pigs at the age of 7–14 weeks. The growth
of diseased pigs is retarded and their feed conversion is lowered, resulting in diminished
production. These studies were designed to investigate the molecular epidemiology of B.
pilosicoli in Finnish sow herds and to improve laboratory diagnostics for B. pilosicoli. Infectivity of the rare hippurate-negative biotype of B. pilosicoli was examined by an infection
trial and eradication of B. pilosicoli from a sow herd was demonstrated.
A high genetic diversity was observed among 131 B. pilosicoli strains obtained from 49
sow herds located in the two major pork production areas of Finland. A high discriminatory power of pulsed-field gel electrophoresis (PFGE) was established with either SmaI or
MluI used as a restriction enzyme. Common genotypes between the herds were rare, and
no clustering of the genotypes according to the two geographical areas was observed. A
single genotype could persist in a herd for several years; however, genetic recombination
among B. pilosicoli strains might occurr. The epidemic nature of B. pilosicoli infection in
Finnish pig farms was shown, and the role of migrating vectors or fomites for horizontal
transmission was assessed as minor.
The high discriminatory power of PFGE was further exploited to investigate hippuratenegative phenotypes of B. pilosicoli. No relationship between hippurate-negativity and
genotypes of B. pilosicoli was detected. This finding was substantiated by comparative
analyses of 16S rDNA nucleotide sequences of hippurate-negative and -positive isolates;
different nucleotide positions of the strains were not predictive of the hippurate hydrolysis reaction. B. pilosicoli isolates from the same herd could possess an identical genome
despite having a different capacity for hydrolysing hippurate. The congruent phylogeny of
hippurate-negative and -positive B. pilosicoli was further ascertained by an ultrastructural
study; the two biotypes shared all of the features unique to the species B. pilosicoli. In
conclusion, the expression of hippurate hydrolysis can vary within a single B. pilosicoli
clone, and thus, the hippurate test can not be used alone for differentiation of porcine B.
pilosicoli.
An occasional hippurate-negativity can disturb the phenotype-based differential diagnostics
of B. pilosicoli. A B. pilosicoli-specific D-ribose test was therefore established to strengthen
the diagnostics protocol. Sixty unrelated B. pilosicoli strains and 35 strains of other porcine
Brachyspira species were studied for D-ribose utilization by an indirect method based on
recording the pH reduction of a broth culture in the presence of D-ribose. All B. pilosicoli
strains, regardless of the hippurate reaction, could utilise D-ribose, whereas the strains
of the other Brachyspira species were D-ribose-negative. These results enabled the construction of an amended classification scheme for phenotypic differentiation of porcine
B. pilosicoli.
Experimental pigs were inoculated either with a hippurate-negative B. pilosicoli strain or
with a B. pilosicoli type strain. Somewhat unexpectedly, the pigs in both trial groups remained healthy. Only the hippurate-negative B. pilosicoli strain could be reisolated from
two of eight infected pigs. An explanation for the silent infection – or lack of infection –
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may lie in the challenge procedure, strain attenuation, environmental conditions, absence
of concomitant enteropathogens or the feeding regimen. The pathogenicity of hippuratenegative B. pilosicoli should be further studied in modified conditions for trial pigs.
Eradication of a chronic B. pilosicoli infection without total depopulation was attempted in
a 60-sow farrowing herd. The principles for eradication of B. hyodysenteriae, the cause of
swine dysentery, were applied. Special attention was paid to sanitary measures, relocation
of animals according to age groups to nearby shelters, husbandry principles and adequate
medication. The eradication was successful; the diarrhoea of the young growers disappeared, and B. pilosicoli was not detected in any sample during the 4.5-year follow-up.
7
List of original publications
This thesis is based on the following original papers, which are referred to in the text by
their Roman numerals. These articles were reprinted with the kind permission of each
journal concerned.
I Fossi M, Pohjanvirta T, and Pelkonen S. 2003. Molecular epidemiological study of
Brachyspira pilosicoli in Finnish sow herds. Epidemiology and Infection. 131: 967–973.
II Fossi M, Pohjanvirta T, Sukura A, Heinikainen S, Lindecrona R, and Pelkonen S. 2004.
Molecular and ultrastructural characterization of porcine hippurate-negative Brachyspira
pilosicoli. Journal of Clinical Microbiology. 42: 3153–3158.
III Fossi M, and Skrzypczak T. 2006. D-ribose utilisation differentiates porcine Brachyspira
pilosicoli from other porcine Brachyspira species. Anaerobe. 12: 110–113.
IV Fossi M, Ahlsten K, Pohjanvirta T, Anttila M, Kokkonen T, Jensen TK, Boye M, Sukura A,
Pelkola K, and Pelkonen S. 2005. Neither hippurate-negative Brachyspira pilosicoli nor
Brachyspira pilosicoli type strain caused diarrhoea in early-weaned pigs by experimental
infection. Acta Veterinaria Scandinavica. 46: 257–267.
V Fossi M, Heinonen M, Pohjanvirta T, Pelkonen S, and Peltoniemi OAT. 2001. Eradication
of endemic Brachyspira pilosicoli infection from a farrowing herd: a case report. Animal
Health Research Reviews. 2: 53–57.
8
Abbreviations
AFLP
ATCC
CVSBA
FA
FCS
FISH
MIC
MLEE
MRP
PCS
PFGE
p.i.
PIS
p.p.m.
RFLP
SD
TEM
TS
WBHIS
amplified fragment length polymorphism
American Type Culture Collection
colistin-, vancomycin- and spectinomycin-containing blood agar
fastidious anaerobe
foetal calf serum
fluorescent in situ hybridization
minimal inhibitory concentration
multilocus enzyme electrophoresis
macrorestriction profile
porcine colonic spirochaetosis
pulsed-field gel electrophoresis
post inoculation
porcine intestinal spirochaetosis
parts per million
restriction fragment length polymorphism
swine dysentery
transmission electron microscopy
trypticase-soy media
weakly β-haemolytic intestinal spirochaete
9
1 Introduction
Brachyspira spp. bacteria are anaerobic, motile spirochaetes that colonize the large intestine of various animal species and humans. Brachyspira pilosicoli belongs to the group of
weakly β-haemolytic intestinal spirochaetes (WBHIS) that causes intestinal spirochaetosis
in pigs, birds and humans (Hampson, 2000). Transmission of B. pilosicoli from animals to
humans has been suggested (Trott et al., 1998), but these zoonotic aspects warrant further
investigations. B. pilosicoli was fully characterized in 1996 (Trott et al., 1996c) but shown
to be pathogenic to pigs much earlier by British researchers (Taylor et al., 1980). In growers
and young fatteners aged 4–20 weeks, B. pilosicoli causes non-fatal, persistent diarrhoea
in a disease known as porcine intestinal spirochaetosis (PIS) (Trott et al., 1996c) or porcine
colonic spirochaetosis (PCS) (Girard et al., 1995). PIS has been recognized worldwide (Duhamel, 1998; Møller et al., 1998; Barcellos et al., 2000b; de Arriba et al., 2002; Choi et al.,
2002 ), in Finland since the 1990s (Heinonen et al., 2000b).
A scheme for phenotypic differentiation of porcine intestinal spirochaetes was developed
in Sweden by Fellström and Gunnarsson (1995). Hippurate hydrolysis capacity was deemed
unique for B. pilosicoli, which made its detection easy. Diagnostic tools for B. pilosicoli soon
expanded to include molecular methods such as polymerase chain reaction (PCR) (Park et
al., 1995; Fellström et al., 1997; Leser et al., 1997), fluorescent in situ hybridization (FISH)
(Boye et al., 1998) and PCR-based restriction fragment length polymorphism (RFLP) (Rohde
et al., 2002).
In the late 1990s, B. pilosicoli was frequently isolated from Finnish pig herds. With the
common use of antimicrobial feed additives, at least 30% of the sow herds were estimated to be carriers of B. pilosicoli (Heinonen et al., 2000b; EELA, unpublished data). The
molecular epidemiological studies on B. pilosicoli performed elsewhere by pulsed-field gel
electrophoresis (PFGE) and multilocus enzyme electrophoresis (MLEE) have shown genetic
heterogeneity among B. pilosicoli strains (Atyeo et al., 1996; Trott et al., 1998; Oxberry and
Hampson, 2003) between and within geographical areas as well as within individual herds.
Similar research in Finland was needed to assess the population structure and potential
infection routes of B. pilosicoli in the Finnish pork production chain.
Phenotypical determination of bacteria species should take into consideration single aberrations in biochemical traits. Biochemically atypical WBHIS bacteria have been isolated
regularly from Finnish pigs since 1997; these isolates have been negative for hippurate
hydrolysis, but according to PCR belong to the species B. pilosicoli, and thus, should be
hippurate-positive. Biochemically aberrant WBHIS isolates have also been described in the
UK (Thomson et al., 2001). Confirmation of phenotyping methods for B. pilosicoli is therefore desired. Utilization of D-ribose has been suggested to be unique to B. pilosicoli within
the genus Brachyspira (Trott et al., 1996c, d). If this proves to be true for a high number of
Brachyspira spp. strains, the test for D-ribose utilization could reinforce the phenotypical
classification of B. pilosicoli.
The pathogenicity of B. hyodysenteriae strains can vary (Jensen and Stanton, 1993; Lee et
al., 1993a; Milner and Sellwood, 1994; Thomson et al., 2003), a phenomenon also suggested apply to B. pilosicoli strains (Duhamel, 1997; Muniappa et al., 1997; Thomson et al.,
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1997; Jensen et al., 2004). A few experiments have attempted to clarify the relationship
between pathogenicity and naturally occurring biochemical or genetic abnormalities in
B. hyodysenteriae and B. pilosicoli; these studies have not demonstrated reduced virulence
(Thomson et al., 2001, 2003). The virulence of hippurate-negative B. pilosicoli isolates was
unknown due to concomitant enteropathogens in the herds of origin. Hippurate-negative
B. pilosicoli has been detected annually in several Finnish pig herds, stressing the need for
studying its pathogenicity.
Chronic B. pilosicoli infection in a herd can be fairly silent, affecting only weight gain of
growers (Duhamel, 1997; Heinonen et al., 2000b; Jacobson et al., 2003; Thomson et al.,
2005). Thereby, B. pilosicoli can be spread by replacement animals and rearing pigs. The
current trend of increased antimicrobial resistance among pathogenic Brachyspira species
(Buller and Hampson, 1994; Molnár et al., 1996; Duhamel et al., 1998a; Karlsson et al.,
2002; Karlsson et al., 2004a, b; Lobová et al., 2004) necessitates attempts to diminish the
prevalence of B. pilosicoli in pig herds. Chronic B. hyodysenteriae infection can be eradicated from farrowing pig herds without total depopulation (Blaha et al., 1987; Christensen et al., 1987; Wallgren, 1988; Ala-Risku, 2005). Eradication of B. pilosicoli has been
assumed, however, to be more difficult. Since B. pilosicoli has a broader host range and
higher resistance to certain physical stress factors outside the host than B. hyodysenteriae
(Trott et al., 1996c), the risk for re-infection has been regarded as high. An eradication
strategy for infectious pig diseases has been widely employed in Finland (Heinonen et al.,
1999, 2000a), and eradication of chronic B. pilosicoli infection was regarded worth of attempt.
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2 Review of the literature
2.1 Taxonomy and general characteristics of intestinal spirochaetes
Brachyspira is the only genus in the family Brachyspiraceae to belong to the order Spirochaetales, together with the families Leptospiraceae and Spirochaetaceae. The order Spirochaetales belongs to the class Spirochaetes. The spirochaetes consist of over 200 species
or phylotypes. Some of these are virulent while others are harmless to the host. Moreover,
many spirochaetes are free-living in the environment. Many spirochaetes are uncultivable;
thus, research of these bacteria is dependent on molecular methods (Paster and Dewhirst,
2000).
All spirochaetes are helical and motile by means of periplasmic flagella. The flagella, with a
count varying from 2 to 100 per cell, are located between the outer sheet and the protoplasmic cylinder, enabling efficient motility of spirochaete cells in a high-viscosity environment. Some spirochaetes are anaerobic, like Brachyspira spp. and Treponema spp., and
some microaerophilic, like Borrelia spp. The species in the genus Leptospira are obligately
aerobic (Canale-Parola, 1984).
Spirochaetes can transform themselves into spheres, a phenomenon reported in certain
species of Borrelia, Leptospira and Brachyspira. Sphere formation is regarded as a response
to deteriorated environmental conditions (Stanton and Lebo, 1988; Faine, 1998; Murgia
and Cinco, 2004).
Figure 1 Cells of Brachyspira pilosicoli, strain Br980 (hippurate-negative phenotype). a. Regularly coiled cell with a typical pointed end. b. Sphere-form of the cell. c. Partially sphere-formed,
comma-shaped cell. Negatively stained with 1% phosphotungstic acid. Bar = 1.0 µm. (Photo:
A. Sukura)
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2.2 Characteristics of Brachyspira spp.
The ecological niche for all recognized Brachyspira species is the lower gastrointestinal
tract, where these spirochaetes are situated close to the mucosal epithelium. Anaerobic
Brachyspira bacteria can metabolize small amounts of oxygen by NADH oxidase, which is
essential for thriving of Brachyspira despite oxygen diffusion from intestinal tissue (Stanton,
1997). This property eases the handling of Brachyspira bacteria in the laboratory, as well.
Brachyspira cells are loosely coiled; their length ranges between 2.0 µm and 14.0 µm,
and their width between 0.19 µm and 0.40 µm. The cell end is blunt, pointed or tapered
depending on the species. The number of periplasmic flagella varies between different
species from 4 to 14; the figures are doubled by overlapping of the flagella in the middle
of the bacterium (Sellwood and Bland, 1997).
Brachyspira bacteria are cultivable on solid and liquid media supplemented with blood or
serum. In liquid media, continuous agitation is a prerequisite for their growth (Stanton and
Lebo, 1988). Selective antimicrobials are needed for isolation of Brachyspira from faecal
or intestinal samples. The colonies are weakly or strongly β-haemolytic depending on the
species. The Brachyspira bacteria can be faintly visualized by Gram-staining. In practice,
the characteristic shape and motility of Brachyspira bacteria are easily seen by, for example,
phase contrast microscope.
2.3 Species in genus Brachyspira
Some Brachyspira species are pathogenic, some are commensals, and pathogenicity of a
few species is unknown or controversial. Seven species of Brachyspira have been characterized to date, and two proposed species are waiting for adequate description. The Brachyspira species and their hosts are presented in Table 1.
Besides the species listed in Table 1, many partially characterized Brachyspira spp. isolates
and Brachyspira-like bacteria in intestinal tissue samples from several animal species need
further identification; Brachyspira-like spirochaetes have been observed in cats (Murray
et al., 2003), opossums (Turek and Meyer, 1979), guinea pigs (Vanrobaeys et al., 1998),
nutrias (Molnár, 1986), horses (Davies and Bingham, 1985; Shibahara et al., 2002), raccoons (Hamir et al., 2001) and Japanese wild deer (Shibahara et al., 2000). Recently, new
phylotypes of Brachyspira spp. have been isolated from several species of wild birds, some
of which can be confused with the well-known species of Brachyspira (Jansson et al., 2001,
2005; Råsbäck et al., 2005).
2.4 Brachyspira pilosicoli in pigs
2.4.1 Specific features of Brachyspira pilosicoli
The B. pilosicoli cell is one of the smallest within the genus Brachyspira; it is 5–12 µm in
length and 0.19–0.30 µm in width. The number of periplasmic flagella varies from 4 to
7, and the flagella are inserted close to the end of bacteria in a single line. The cell end of
14
Table 1 The species in genus Brachyspira
Species1
Host
Disease
Reference
B. hyodysenteriae
Pig
Swine dysentery
Harris et al., 1972
Rhea
Typhlocolitis
Jensen et al., 1996
Mallard
Not reported
Jansson et al,. 2004
Pig
Controversial
Stanton et al., 1997;
Binek and Szynkiewicz, 1984
Poultry
Intestinal spirochaetosis
Griffiths et al., 1987;
McLaren et al., 1997
B. innocens
Pig
Not reported
Kinyon and Harris 1979;
Trott et al., 1996a
B. murdochii
Pig
Controversial
Stanton et al., 1997;
Jensen et al., 2005;
Hampson et al., 1999
B. pilosicoli
Pig
Porcine intestinal
spirochaetosis
Trott et al., 1996c
Poultry
Intestinal spirochaetosis
Stephens and Hampson, 2002;
Shivaprasad and Duhamel, 2005
Man
Intestinal spirochaetosis
Trivett-Moore et al., 1998;
Jensen et al., 2001
Dog
Intestinal spirochaetosis
Duhamel et al.,1998b;
Fellström et al., 2001a
B. alvinipulli
Poultry
Intestinal spirochaetosis
Swayne et al., 1995;
Stanton et al., 1998
B. aalborgi
Man
Intestinal spirochaetosis
Hovind-Hougen et al., 1982
Non-human
primate
Intestinal spirochaetosis
Duhamel et al., 1997;
Munshi et al., 2003
“B. canis”
Dog
Not reported
Duhamel et al., 1998b;
Johansson et al., 2004
“B. pulli”
Poultry
Not reported
McLaren et al., 1997;
Phillips and Hampson, 2005
B. intermedia
1 The proposed new species are indicated in quotation marks.
B. pilosicoli is distinctively pointed and covered by a unique lattice-like structure. This structure has been proposed to be important in pathogenesis (Sellwood and Bland, 1997).
B. pilosicoli is weakly β-haemolytic on blood agar. Like most of the other weakly β-haemolytic intestinal spirochaetes (WBHIS), B. pilosicoli grows well on the surface of agar, whereas
the strongly β-haemolytic B. hyodysenteriae, the cause of swine dysentery (SD), tends to
also grow below the agar surface. B. pilosicoli grows relatively fast in optimal conditions
at 37–42°C; its doubling time, 1–2 h, is about half of that of other porcine Brachyspira
species. B. pilosicoli tolerates oxygen slightly better (7% vs. 5% in headspace of broth
15
culture) and grows in a wider pH range (pH 5.6–8.0 vs. pH 6.0–8.0) than the other porcine
Brachyspira species (Trott et al., 1996c). B. pilosicoli survives outside the host longer than B.
hyodysenteriae. In laboratory conditions, B. pilosicoli has survived in faeces over 210 days
and in soil or faeces-mixed soil over 119 days at 10°C (Boye et al., 2001). In lake water, B.
pilosicoli has survived 66 days at 4°C and 4 days at 25°C (Oxberry et al., 1998).
The pattern of antibiotic susceptibility is important to consider when a selective medium
for isolation is formulated. Like B. hyodysenteriae, B. pilosicoli is resistant to spectinomycin,
colistin and vancomycin. However, spiramycin and rifampin retard the growth of B. pilosicoli, whereas B. hyodysenteriae is resistant to these antimicrobicials (Trott et al., 1996c).
2.4.2 Porcine intestinal spirochaetosis
PIS has been recognized worldwide in all major pork-producing areas. In studies conducted
in some European countries and Brazil, B. pilosicoli has been isolated from 1–52% of investigated herds (Møller et al., 1998; Thomson et al., 1998; Barcellos et al., 2000b; Heinonen
et al., 2000b; de Arriba et al., 2002; Jacobson et al. 2005). The results of these studies are
not, however, comparable because some of the surveys have targeted diarrhoeic herds,
and some have examined both healthy and diarrhoeic herds. B. pilosicoli was detected in
only one out of 125 Spanish herds with diarrhoea problems (de Arriba et al., 2002). The
routine use of antimicrobials might have suppressed the growth of B. pilosicoli in some
herds, thereby masking its presence. Additionally, the use of antimicrobial growth promoters in feed might have concealed B. pilosicoli positive herds. This was suggested by Heinonen et al. (2000b), who investigated the regional prevalence of intestinal spirochaetes
in Finland by random sampling of the herds; B. pilosicoli was isolated from only one out of
20 herds using carbadox as a growth promoter, whereas B. pilosicoli was detected in three
out of four herds not using antimicrobial feed additives.
PIS most commonly affects pigs aged 8–13 weeks, but symptoms are sometimes observed
at 5–20 weeks of age, as well. The incubation time varies from 2 to 20 days. The infection
causes typhlocolitis, which manifests as loose, cement-like faeces with occasional blood
flecks. Pigs are non-febrile or their body temperature is slightly raised. Weight gain of pigs
is markedly reduced during the diarrhoeic period. Recovery time varies, and the disease
leads to uneven growth of pigs in the same group. Mortality is low unless intestinal coinfections aggravate the lesions (Taylor and Trott, 1997; Thomson et al. 1998; Hampson
and Trott, 1999).
The gross lesions on the mucosa of the large intestine vary from almost normal to increased
mucus excretion, oedema, haemorrhage and focal necrosis. Microscopically, a neutrophilic
and lymphocytic typhlocolitis with microabscesses can be seen. B. pilosicoli cells attach
onto the epithelial surface densely and side by side, which causes a phenomenon known as
a false brush-border. The only other Brachyspira species capable of forming a similar fringe
of densely packed spirochaetes on the intestinal epithelium is B. aalborgi, which is encountered in humans and non-human primates. This phenomenon is seemingly dependent on
the stage of infection and the degree of post-mortem changes because it is rarely seen
in routine necropsy material (Taylor and Trott, 1997; Thomson et al., 1997; Jensen et al.,
2004). Beside epithelial attachment, B. pilosicoli can invade the intestinal lamina propria
(Jensen et al., 2000; Duhamel, 2001).
16
b
Figure 2 a. A crypt of colonic mucosa from a pig affected by porcine intestinal spirochaetosis.
Spirochaetes in cryptal lumen (*). Bar = 50 µm. b. Spirochaetes invading the crypt epithelium.
Magnified from Figure A. Bar = 10 µm. Warthin-Starry stain. (Photo: M. Fossi)
Specific virulence factors of B. pilosicoli remain poorly understood. One explanation for
the diarrhoea is that the densely packed spirochaetes on epithelial cells cause a physical
blockage of absorption of fluids and disrupt the epithelial microvilli (Taylor and Trott, 1997).
Recent studies have investigated proteolytic membrane proteins of B. pilosicoli that may
interact with the host (Trott et al., 2004, Dassanayake et al., 2004).
The severity of PIS depends on several factors. Managemental measures for diminishing
the amount of circulating bacteria are of great importance. Co-infections with other enteric pathogens, especially with Lawsonia intracellularis, Yersinia spp. or Salmonella spp.,
aggravate the symptoms (Thomson et al., 1998; Jacobson et al., 2003). Variability in pathogenicity of different B. pilosicoli strains (Thomson et al., 1997; Jensen et al., 2004) and individual differences in hosts’ cellular responses observed in conjunction with SD (Jonasson
et al., 2004), may also have an influence on manifestation of PIS.
2.4.3 Detection of Brachyspira pilosicoli
The diagnosis for PIS is based on detection of the organism by culture or molecular methods.
Because B. pilosicoli strains are antigenically highly diverse (Lee and Hampson, 1999), no
serological methods for diagnosis are yet available.
2.4.3.1 Isolation methods
Rectal samples from live pigs should be placed in transportable media intended for anaerobes. For transport, the samples should be cooled but not frozen. Several combinations of antimicrobial agents are used for selective culture of Brachyspira spp. CVSBA is a
commonly used solid medium containing colistin, vancomycin and spectinomycin, and it
is suitable for searching for all porcine Brachyspira species (Jenkinson and Wingar, 1981).
Some media that have been developed for identifying B. hyodysenteriae include spiramycin
17
and rifampin, and therefore are suboptimal for isolation of B. pilosicoli (Kunkle and Kinyon,
1988). Enrichment methods have been developed for isolating B. hyodysenteriae (Fellström
et al., 2001b) and B. pilosicoli (Calderaro et al., 2005), but in the case of B. pilosicoli further
comparative studies between the methods are needed.
Isolation of Brachyspira spp. from faecal or intestinal samples is enhanced when the agar
surface has been sliced before culturing. The spirochaetes glide along the cuts out of the
cultivation site, and thus, a pure subculture is achieved faster (Olson, 1996). According to
our unpublished studies, the speed of diverging growth of Brachyspira is enhanced when
the cuts on the agar are very shallow. Further subcultures on nutrient-rich media, such as
fastidious anaerobe agar (FA), provide a sufficient bacteria mass for subsequent analyses.
The strains are stored at −70°C, or better yet in liquid nitrogen (−196°C), in a broth containing serum and glycerol.
2.4.3.2 Phenotypic differentiation
Differential diagnostics of porcine B. pilosicoli by biochemical methods is based on positivity for hippurate hydrolysis and negativity for β-glucosidase (Fellström et al., 1995). Hip-
b
Figure 3 Growth of B. pilosicoli from porcine faecal sample (a) streaked transversally on presliced selective CVSBA plate. Three-day-old culture incubated anaerobically at 42°C. Diverging
growth of B. pilosicoli has reached the ends of the cuts (b). (Photo: M. Fossi)
18
purate and glucosidase tests are simple 4-h or overnight tests, which record the indicative
colour change in test broths that have been incubated aerobically at 37°C. The hippurate
test has been proposed as a key test for B. pilosicoli, offering good species determination
(Fellström et al., 1997). To date, avian B. alvinipulli is the only other Brachyspira species
that has been shown to hydrolyse hippurate (Stanton et al., 1998). Glucosidase tests may
be overlooked because of the distinctive hippurate test for B. pilosicoli. In our experience,
the results from glucosidase tests are, however, sometimes poorly interpretable because of
a faint colour reaction.
Occasional biochemically aberrant strains, or mixed cultures of several Brachyspira strains
can confuse species determination. In such cases, the presence of B. pilosicoli can be suggested by microscopy; the reference strains of B. pilosicoli and B. hyodysenteriae as well
as the culture being studied are smeared side by side on the same slide for comparative
evaluation of bacteria cell sizes.
Tests for carbohydrate utilization are not in common use in Brachyspira diagnostics. Utilization of various sugars has been studied only for a limited number of Brachyspira strains,
mainly in conjunction with characterization of the type strains of Brachyspira species (Trott
et al., 1996d; Stanton et al., 1997, 1998). Carbohydrate utilization tests last at least overnight and require anaerobic conditions, which might explain their disfavour.
2.4.3.3 Molecular identification
Numerous DNA techniques have been applied for taxonomic and subspecies resolution
of Brachyspira bacteria. Sequence analysis of genes coding for ribosomal RNA (rDNA) has
been adopted as a powerful tool for phylogenetic studies of bacteria (Olsen and Woese,
1993). The bacterial 16S rDNA is highly conserved in general, and similarity of 16S rDNA
sequences among the Brachyspira species is reported to be ≥ 95.7% (Pettersson et al.,
1996). The sequencing of 16S rDNA in particular has resolved the taxonomy of Brachyspira
spp. from genus to subspecies level (Pettersson et al., 1996, 2000; Johansson et al., 2004).
In 16S rDNA, there are eight evolutionary, highly conserved universal sequences, which
are exploited as landmarks when the areas between the universal sequences are studied.
B. pilosicoli has a unique hexamere of consecutive thymines in 16S rDNA nucleotide positions 176–181 (consensus sequence of Brachyspira spp., Pettersson et al., 1996), an area
commonly included in the forward primer for several B. pilosicoli-specific polymerase chain
reaction (PCR) applications (Park et al., 1995; Fellström et al., 1997; Mikosza et al., 2001).
However, a hexamere and even a heptamere of thymines have been identified in 16S rDNA
sequences of various avian Brachyspira isolates other than B. pilosicoli just recently (Jansson et al., 2005). These strains cause false diagnosis when only a 16S rDNA-based PCR is
used.
Leser et al. (1997) developed a B. pilosicoli-specific PCR based on 23S rDNA. Used in conjunction with a 16S rDNA-based PCR, the risk of false results due to a single nucleotide
aberration can be avoided. The NADH oxidase gene (nox) has been shown to be a suitable target for PCR applications for several porcine Brachyspira species, but in the case of
B. pilosicoli its sensitivity is poor (Atyeo et al., 1999b). A PCR-based restriction fragment
length polymorphism (RFLP) has been applied for differentiation of Brachyspira species
19
from porcine (Rohde et al., 2002) or avian (Townsend et al., 2005) origin. The restriction
patterns obtained with RFLP are species-specific, and the agreement between RFLP and
species-specific PCR is quite good (Townsend et al., 2005).
A species of Brachyspira can be identified from fixed tissue samples by PCR when the
specific target region in the DNA is relatively short, preferably less than 500 bp in length.
Kraatz et al. (2001) identified B. pilosicoli and B. aalborgi from human colonic biopsies by
sequencing the 207-bp amplicons from 16S rDNA obtained by primers designed for the
genus Brachyspira. Mikosza et al. (1999) approached the same issue, but focused specifically on detection of B. pilosicoli and B. aalborgi from the biopsies; the species-specific
primers designed for 196- to 472-bp sequences from both 16S rDNA and nox yielded the
expected amplicons.
Fluorescent in situ hybridization (FISH) is a method in which an oligonucleotide probe is
used for visualization of the target organism in tissue. This is advantageous when exact
localization of the organism in tissue is needed or when the organism is unculturable. The
specificity of designed probes can vary between domain bacteria and a single strain. For
Brachyspira research, probes targeting 16S or 23S rDNA have been developed for B. pilosicoli, B. hyodysenteriae and B. aalborgi, as well as for the genus Brachyspira spp. (Boye et
al., 1998; Jensen et al., 2001, 2005). In routine work, FISH is a faster method for in situ
detection than e.g. immunohistochemical methods, but post-mortem autolysis of tissue
samples can cause false-negative results. For B. pilosicoli, the sensitivity of FISH does not
appear to exceed the sensitivity of cultivation, probably due to the localization of B. pilosicoli bacteria on the surface of the epithelium (Jensen, 2005).
2.4.3.4 Fingerprinting
For epidemiological purposes, subspecies differentiation of Brachyspira spp. is typically done
by multilocus enzyme electrophoresis (MLEE) (Lee et al., 1993b; Stanton et al., 1996; Trott
et al., 1996a, 1998; Oxberry et al., 2003), pulsed-field gel electrophoresis (PFGE) (Atyeo
et al., 1996, 1999a; Trott et al. 1996a, 1998; Rayment et al., 1997; Atyeo et al., 1999a;
Fellström et al., 1999; Suriyaarachichi et al., 2000), or by amplified fragment length polymorphism (AFLP) (Møller et al., 1999).
MLEE is one of the most frequently used methods for typing of intestinal spirochaetes. It
has been used for identifying all of the currently recognized Brachyspira species, as well
as for within-species subtyping. MLEE is, however, too time-consuming and laborious for
daily use. PFGE is a popular method for subspecies typing, not only because of its high discrimination power but also because of its excellent reproducibility. The first comprehensive
study of subspecies differentiation of B. pilosicoli strains was done by PFGE (Atyeo et al.,
1996). The discriminatory power was found to be superior to MLEE, a finding confirmed
in subsequent studies (Rayment et al., 1997; Trott et al., 1998). Recent technical improvements have decreased the drawbacks of PFGE, i.e. time-consuming protocol and occasional DNA degradation (Gautom, 1997; Koort et al., 2002). The third technique, AFLP, has
been found to have a discriminatory power equal to that of PFGE, but its reproducibility is
slightly lower (Møller et al., 1999; Savelkoul et al., 1999).
20
2.5 Transmission of infection
In a herd, PIS can spread by direct pig contact, by feed, by faeces-contaminated surfaces
and fomites and by water. Replacement breeding animals and rearing pigs can transmit
the infection between herds (Taylor and Trott, 1997; Oxberry et al., 1998, 2003). In addition, evidence has emerged that wild birds and mice can act as reservoirs and transmit
B. pilosicoli to pigs (Oxberry et al., 1998; Fellström et al., 2004). A natural cross-species
transmission of B. pilosicoli to pigs has not been confirmed, but an experimental infection
of weaned pigs by human B. pilosicoli strains led to symptoms of PIS (Trott et al., 1996b).
Moreover, an experimental infection of chickens by porcine and human B. pilosicoli strains
caused avian intestinal spirochaetosis with symptoms (Trott et al., 1995).
Transmission of intestinal spirochaetes from nursing sows to offspring has not been reported. Young sucklings probably can not be colonized by anaerobic spirochaetes because
of the higher oxygen concentration in their intestines than in weaned pigs (Hillman et al.,
1993) and because of protective antibodies in sow’s milk. The creep feed served before
weaning might increase fermentation and subsequently decrease oxygen content in the
colon, thus enabling colonization by intestinal spirochaetes.
2.6 Treatment and control of porcine intestinal spirochaetosis
Clinical PIS can be treated by antimicrobials. Macrolides, such as tylosine and lincomycin,
and tiamulin and valnemulin, belonging to pleuromutilins, have been among the most
popular agents used to treat spirochaetal diarrhoea in pigs. The medication should, however, be combined with improved hygiene in the pens and prevention of carriage by fomites. Reappearance of symptoms after medication is discontinued is common (Spearman
et al., 1988), especially when improved cleaning measures are not permanently adopted.
Antimicrobial resistance of Brachyspira spp. has become common, especially among B.
hyodysenteriae strains, but the trend has also been observed in B. pilosicoli strains (Duhamel et al., 1998a; Fossi et al., 1999; Karlsson et al., 2004b). This can be explained by
the frequent co-occurrence of B. pilosicoli and B. hyodysenteriae, with B. hyodysenteriae
being the principal initiator of therapy, as well as by continuous low-level medication on
farms (Thomson et al., 1998; Stege et al., 2000). Trans-species spread of genes responsible
for antimicrobial resistance among Brachyspira spp. has not been reported. Pleuromutilins
have been effective against porcine spirochaetal diarrhoea (Blaha et al., 1987; Duhamel et
al., 1998a; Messier et al. 1990). However, the minimal inhibitory concentration (MIC) values of pleuromutilins have been elevated for an increasing proportion of B. hyodysenteriae
and B. pilosicoli isolates investigated over the last decade (Molnár, 1996; Karlsson et al.,
2004a, b; Lobová et al., 2004).
Good management principles, segregation of pigs by age group, and sanitation measures,
including rodent control, are crucial preventative measures for PIS. B. pilosicoli is sensitive
to most of the common disinfectants (Corona-Barrera et al., 2004) and to drying. At a
temperature of −70°C, B. pilosicoli survived in faeces for 14 days (Barcellos et al., 2000a).
Similar studies at temperatures of 0°C to −20°C, simulating winter in northern areas, are
21
lacking. In our experience, recovery of Brachyspira spp. from faecal samples after a short
freezing period at −18°C to −20°C is very poor.
The prevalence and severity of spirochaete-induced diarrhoea in pigs are feed-related (Durmic et al., 1998; Pluske et al., 1998; Stege et al., 2001). Generally, fermentable carbohydrates in the large intestine promote thriving of intestinal spirochaetes. Experimentally, a
highly digestible diet based on cooked rice and animal protein has prevented colonization
of B. hyodysenteriae (Pluske et al., 1996). In a corresponding study for B. pilosicoli, colonization of bacteria could not be prevented, but the symptoms of PIS were milder (Hampson
et al., 2000). From an economic point of view, highly digestible feed is not feasible in
practice.
To date, no effective vaccine for control of PIS exists. One vaccination trial investigating
parenteral B. pilosicoli bacterin has been reported. When infected with the homologous
B. pilosicoli strain, vaccinated and unvaccinated pigs developed PIS with equal intensity
(Hampson et al., 2000). A recent immunization trial for SD using a subunit vaccine from recombinant outer-membrane lipoprotein BmpB of B. hyodysenteriae has been promising (La
et al., 2004). To our knowledge, no similar studies with B. pilosicoli have been published.
22
3 Aims of the study
The aims of the thesis were to investigate epidemiological characteristics of porcine B.
pilosicoli and to improve differential diagnostics of the infection. The specific aims were as
follows:
1 to study the genetic diversity of B. pilosicoli in Finnish pig herds (I, II);
2 to investigate the genetic relationship between hippurate-negative and -positive phenotypes of B. pilosicoli (II), and to reinforce the scheme for phenotypical differentiation
of porcine B. pilosicoli by adding a discriminating carbohydrate utilization test (III);
3 to investigate the infectivity of hippurate-negative B. pilosicoli by experimental infection (IV);
4 to study the possibility of eradicating B. pilosicoli infection from a sow herd without
total depopulation (V).
23
4 Materials and methods
4.1 Sampling (IV, V)
The faecal samples from live pigs were taken by swabs which were placed into transportable culture media intended for facultative and obligatory anaerobes (Probact transport
swab, Technical Service Consultants Ltd., Heywood, UK). The swabs were streaked on
culture media within 48 h (V) or 4 h (IV). At necropsy (IV), pieces 5–10 cm in length were
dissected from the caecum, the proximal, mid-colon and distal colon, and from the rectum.
The intestine samples were transsected just prior to cultivation, and the mucosal scrapings
were cultured within 4 h of necropsy.
4.2 Bacterial isolates and strains (I–V)
For the molecular epidemiological study (I), altogether 131 B. pilosicoli isolates were chosen
from isolates obtained during 1997–2000 from 49 farrowing and farrow-to-finish herds
located in the southern (S) and northern (N) pork production areas of Finland. Diarrhoea
was a problem in 38 herds, no diarrhoea was observed in five herds and health status was
unknown in six herds. In 41 herds, 1–10 B. pilosicoli isolates per herd were obtained from
rectal samples of weaned pigs. In eight herds, a single B. pilosicoli isolate from necropsy
samples was included.
For characterization of hippurate-negative B. pilosicoli (B. pilosicoli hipp-) (II), 11 B. pilosicoli hipp- isolates were studied. The isolates were obtained during 1997–2000 from seven
unrelated farrowing and farrow-to-finish herds. In addition, nine biochemically common B.
pilosicoli isolates, obtained concomitantly with B. pilosicoli hipp- isolates from three of these
herds, were included.
For the study of D-ribose utilization (III), 53 B. pilosicoli isolates with a common biochemistry,
seven hippurate-negative biovariants of B. pilosicoli, ten B. hyodysenteriae, eight B. intermedia and 14 Brachyspira spp. group III isolates were used. The isolates were obtained
during 1997–2004. Thirty-six of the B. pilosicoli isolates had been shown to represent different genotypes (in Study I), and seven of the B. hyodysenteriae isolates to have dissimilar
lipo-oligosaccharide profiles in their cell walls (Fossi et al., 2004). The remaining isolates
were obtained from unrelated sources.
The B. pilosicoli hipp- strain Br1622 for the infection trial (IV) originated from a farrowing
herd sampled in 2000. The 16S rDNA sequence of this strain possessed 12 nucleotide mismatches with the respective sequence from B. pilosicoli type strain P43/6/78T.
For the eradication study (V), weaners of the trial herd were sampled before eradication
four times between January and May 1997. At least one B. pilosicoli isolate per sampling
(altogether six isolates) was stored. Two and five B. innocens isolates, before and after
eradication, respectively, were also stored.
The type and reference strains used in these works were purchased from the American
Type Culture Collection (ATCC): B. pilosicoli P43/6/78T (ATCC 51139) (I–III), B. hyodysente-
24
riae B78T (ATCC 27164) (II), B. hyodysenteriae B204 (ATCC 31212) (II, III), B. intermedia
PWS/AT (ATCC 51140) (III), B. innocens B256T (ATCC 29796) (III) and B. murdochii 155–20
(ATCC 700173) (III). All of the isolates were stored at −70°C in beef broth (Merck, Darmstadt, Germany) supplemented with 12% horse serum and 15% glycerol.
4.3 Isolation (IV, V) and phenotypic identification (I–V)
Faecal swabs and intestinal scrapings were streaked on pre-reduced and pre-sliced selective
CVSBA plates transversally over the agar cuts. For selection, the agar base (Blood agar base
no 2, Oxoid Ltd., Basingstoke, Hampshire, England) with 5% defibrinated sheep blood and
1% sodium ribonucleinate was supplemented with 0.4 mg ml −1 spectinomycin, 0.025 mg
ml −1 colistin and 0.025 mg ml −1 vancomycin. The plates were incubated in anaerobic chambers at 42°C in an atmosphere of 90% N 2 and 10% CO2 (AnaerogenTM, Oxoid Unipath
Ltd.). The plates were inspected every third or fourth day up to 14 days for Brachyspira-like
growth. Suspected Brachyspira colonies were subcultivated on CVSBA until the culture was
deemed pure by microscopy. For species determination by biochemical methods and for
storage, the isolates were subcultured on fastidious anaerobe (FA) agar (Lab MTM, Lancashire, UK), which was supplemented with 7–10% defibrinated horse or bovine blood.
The Brachyspira isolates were phenotyped according to the scheme of Fellström et al.
(1999). Haemolysis was assessed as weak or strong by culturing the isolate, the B. hyodysenteriae reference strain B204, and the B. pilosicoli type strain P43/6/78T simultaneously
on the same typticase-soy (TS) agar (BBLTM, Becton, Dickinson and Co., Le Pont de Claix,
France) plate supplemented with 5% defibrinated bovine blood.
Indole production was assessed by smearing the culture on a filter paper saturated with a
reagent containing 1% p-dimethylaminocinnamaldehyde in 10% hydrochloric acid (Sutter
and Carter, 1972). Appearance of a bluish-purple colour indicated positive reaction, and a
pink tone a negative reaction for indole production. B. intermedia PWS/AT was used as a
positive control. The test for hippurate hydrolysis was performed according to Rübsamen
and Rübsamen (1986) and is described in detail in Paper III. The tests for α-galactosidae, αglucosidase and β-glucosidase activities were performed by using the respective diagnostic
tablets (DiatabsTM, Rosco Diagnostica A/S, Taastrup, Denmark) according to the manufacturer’s instructions.
Isolates showing weak β-haemolysis, positive hippurate reaction, negative indole reaction
and negative α-glucosidase and β-glucosidase reactions were classified as B. pilosicoli. The
strains with the reactions listed above except for no positive hippurate reaction were preliminarily assessed as atypical isolates. For seven atypical isolates, which originated from
different herds, the stability of the negative hippurate reaction was confirmed as follows:
consecutive culture passages, which were incubated in parallel at 38°C and 42°C as doublets on FA agar, were tested for hippurate hydrolysis from seven to ten passages. All of the
test results were negative for hippurate hydrolysis. All of these isolates were included in
Study II, and one of the isolates in Study IV.
The protocol for the D-ribose test is described in detail in Paper IV. In brief, D-ribose utilization was demonstrated indirectly by recording pH reduction in the broth culture. Tryptonepeptone base broth contained 7% foetal calf serum (FCS). One per cent D-ribose was
25
added for testing D-ribose utilization. A sugar-free base broth was added on each testing
occasion. The bacteria were suspended in the broths and incubated anaerobically on a
planar shaker for 28–30 h. The difference in pH reduction between the D-ribose broth and
the base broth was recorded. One per cent glucose or mannose replaced D-ribose when
utilization of these sugars was tested. Reproducibility of the test and optimal cell density
in broths were evaluated as follows: two B. pilosicoli field strains and the B. pilosicoli type
strain P43/6/78T were tested three times in D-ribose broth supplemented with FCS, in parallel in twofold cell densities of 2.3 × 107, 4.5 × 107 and 9.0 × 107 cells ml −1, equivalent to
McFarland units 2, 4 and 8, respectively. The reduction in pH was compared between the
different cell densities in tests, and variation in pH reduction between test occasions in different cell densities was analysed.
4.4 Polymerase chain reaction (I–V)
All of the isolates were studied by two B. pilosicoli-specific PCR assays. The primers targeting 930 bp of ribosomal 16S rDNA have been described by Fellström et al. (1997), and
those for 143 bp of 23S rDNA by Leser et al. (1997). The PCRs were performed as outlined
by these authors.
A species-specific nested-PCR was used for detection of L. intracellularis in Study IV. The
samples were prepared according to McOrist et al. (1994), and the primer pairs and PCR
protocol described by Jones et al. (1993) were applied.
4.5 Sequencing of 16S rDNA (III)
The nucleotide sequence of 16S rDNA was almost completely sequenced for three B. pilosicoli hipp- isolates. In addition, a partial 16S rDNA sequence of 877 bp was studied for five B.
pilosicoli hipp- isolates and two B. pilosicoli isolates. An almost complete 16S rDNA sequence
(1435 bp out of 1485 bp) was obtained as described by L’Abee-Lund et al. (2003). All of
the primers were universal (Weisburg et al., 1991). The nucleotide sequence obtained,
with a length of 1435 bp, covered the Brachyspira consensus sequence (Pettersson et al.,
1996) from position −8 to position 1431.
The partial 16S rDNA sequence of 877 bp, covering the Brachyspira consensus sequence
from position 183 to position 1059, was obtained from the amplicon produced by B.
pilosicoli-specific PCR. The PCR was performed as described by Fellström et al. (1997). The
amplicon was sequenced directly or cloned with a PCR-Script Amp cloning kit (Stratagene,
La Jolla, CA, USA) according to the manufacturer’s instructions. The sequencing was purchased from AIV Institute, Kuopio, Finland.
4.6 Pulsed-field gel electrophoresis (I, II, IV, V)
Preparation of agar plugs, macrorestriction of bacterial DNA and electrophoresis are described in detail in Paper I. In brief, the washed bacteria in phosphate-buffered saline were
mixed in agarose and hardened in a mould. The agarose plugs were placed in lysis solution
26
containing lysozyme and RNAase for 24 h at 37°C. Next, the plugs were placed in proteinase solution (proteinase K, sodium lauroyl sarcosine) for 24 h at 50°C. Prior to digestion,
the plug slices were dialysed in TE buffer. Rare-cutting enzyme MluI was used for macrorestrictive digestion of all isolates and strains, and SmaI was used for 70 out of 131 isolates
or strains in Study I as well as for all 13 isolates in Study V.
The digested plugs were loaded on to a 1% agarose gel and subjected to electrophoresis
for 22 h at 14°C with a linear ramp of 2–30 s and 9–55 s for plugs digested with MluI
and SmaI, respectively. The gels supplemented with ethidium bromide were photographed
with UV illumination, and the images were scanned and analysed using GelCompar program (Applied Maths, Kortrijk, Belgium). Clustering analysis, based on the unweighted
pair-group method with arithmetic averages (UPGMA), was performed in the first and
second studies.
4.7 Fluorescent in situ hybridization (IV)
The colon and caecum sections of all 23 pigs in the infection trial were subjected to fluorescent in situ hybridization (FISH). The oligonucleotide probes used are described in Paper IV.
A species-specific probe for B. pilosicoli hipp- strain Br1622 and genus-specific probes for
Treponema, Leptospira and Borrelia were selected using ARB software (Strunk et al., 2000).
Br1622 could be differentiated from B. pilosicoli type strain P43/6/78T because of differences in 16S rDNA nucleotide sequences. The probe designed for Br1622 had at least two
mismatches to all other Brachyspira species in the database. The general bacterial probe
EUB338, the genus-specific probe for Brachyspira and the species-specific probe for B.
pilosicoli have been described previously by Boye et al. (1998).
Prior to hybridization, the sections were deparaffinized in xylene and transferred to ethanol
for 10 min. The sections were circumscribed with a hydrophobic PAP pen (Daido Sangyo
Co. Ltd., Tokyo, Japan). Hybridization was carried out at 37°C for 16 h in a moisture chamber with 30 µl of hybridization buffer (100 mM Tris, 0.9 M NaCl, 0.1% sodium dodecyl
sulphate) containing 150 ng of probe. Washing was done in 100 ml of hybridization buffer
for 15 min and then in 100 ml of washing solution (100 mM Tris, 0.9 M NaCl) for 15 min.
The samples were rinsed in water, air-dried and mounted in Vectashield (Vector Laboratories Inc., Burlingame, CA, USA) prior to microscopy.
An Axioplan2 epifluorescent microscope (Carl Zeiss, Oberkochen, Germany) equipped with
a 75 W Xenon lamp and filter set XF53 (Omega Optical, Brattleboro, VT, USA) was used for
simultaneous detection of red and green fluorescence.
4.8 Transmission electron microscopy (II, IV)
For ultrastructural study of cultured bacteria, B. pilosicoli hipp- isolates Br980 and Br1048,
originating from different herds, B. hyodysenteriae type strain B-78T and B. pilosicoli type
strain P43/6/78T were propagated on FA agar for three days at 37°C. The bacteria were
suspended in 0.1 M phosphate buffer at a concentration of 1.0 × 107–8 cells ml −1. The suspension was applied to carbon-coated grids and incubated for 1 min. The grid was then
27
rinsed with distilled water. Finally, the samples were negatively stained with 1% phosphotungstic acid for 10–20 min (Utriainen et al., 1997).
For ultrastructural study of unidentified bacteria in situ, 1.0 mm2 samples of the colonic
and caecal mucosa were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 4 h.
The samples were embedded in EponTM, and thin sections were stained with 3% uranyl
acetate for 40 min and 3% lead citrate for 1.5 min.
The samples were visualized and photographed using a Jeol Jem 100-s electron microscope. For the cultured Brachyspira bacteria, mean values for the cell dimensions were
calculated from the measurements of 20 bacteria in the images.
4.9 Antimicrobial susceptibility testing (V)
Sensitivity of three B. pilosicoli and two B. innocens isolates to tiamulin before eradication, and three B. innocens isolates after eradication was studied. The MIC of tiamulin was
determined by an agar dilution method using twofold dilutions of tiamulin-fumarate (Bio
Cheme®) from 1.0 µg ml −1 to 0.063 µg ml −1 in TS agar supplemented with 5% defibrinated
sheep blood (Rønne and Szancer, 1990; Fossi et al., 1999). Plastic plates of 9 cm diameter
were used (Sterilin Ltd., code 101RT). TS agar without tiamulin was included in each assay
for growth control.
The bacteria culture, which was grown on FA agar, was suspended in buffered saline,
and the density of bacteria was adjusted with the barium sulphate standard of McFarland
no.1 (bioMèrieux®). Three droplets of bacteria suspension were spread on each test and
control agar. The agar plates were incubated for six days anaerobically at 42°C. The lowest
concentration of tiamulin that prevented haemolytic growth was interpreted as the MIC. A
strain was assessed as sensitive to tiamulin when MIC was ≤ 1 µg ml −1 (Rønne and Szancer,
1990).
4.10 Study design for hippurate-negative B. pilosicoli
infection trial (IV)
Details of study design and measures during the trial are provided in Study V. In brief, the
24 piglets were separated from their sows at the age of 11 days and moved to barrier
facilities, where they were raised together until being divided into three trial groups at the
age of 45 days. During the next two days, the pigs in groups 1 and 2 were inoculated intragastrically with doses of 3.2–4.0 × 109 cells of B. pilosicoli hipp- strain Br1622 and B. pilosicoli
type strain P43/6/78T, respectively. The third trial group was sham-inoculated with sterile
broth. Faecal samples for enteric pathogens and blood samples for haematology were
taken, and the body weights were recorded regularly. The pigs were necropsied within
7–23 days post inoculation (p.i.), and samples were taken for routine histopathology, TEM
and microbiology. Antibodies for L. intracellularis were analysed by using an indirect fluorescent antibody test (IFAT) (McOrist et al., 1987; Lawson et al., 1988; Knittel et al., 1998)
from the colostrum of sows and the sera of pigs at the age of 42 days and at necropsy.
28
4.11 Actions taken for eradication of B. pilosicoli (V)
The farrowing herd of 60 sows had suffered from post-weaning problems for many years.
All four samplings between January and August 1997 had revealed B. pilosicoli in abundance. The MIC values of tiamulin for the three B. pilosicoli isolates analysed were < 0.125
µg ml −1.
Eradication was undertaken during August-October 1997. Before moving the animals,
in-feed medication with tiamulin (Tiamutin®) 200 p.p.m. was started. The sucklings were
not medicated. The sows were medicated for 30 days, the weaners for 23 days and the
finishing pigs for 18 days.
The old, non-segregated piggery had to be emptied at the same time; the farrowing sows
and weaners were moved to a barn 20 m from the piggery, the dry sows and the boar to a
paddock next to the piggery, and the finishing pigs to a shed located 100 m from the piggery. None of the growing pigs were returned to the piggery but were sold by December
1997.
The piggery was swept, washed, disinfected and left empty for 25 days. Old wooden materials were burned and worn surfaces repaired. The manure pit was disinfected and the
manure was transported to remote fields. Rodents were controlled during and after the
eradication by poison baits.
The sows and the boar were returned to the piggery between September 10 and October
3. All replacement animals bought after this were medicated with Tiamutin® 200 p.p.m.
for three weeks, during which time they were quarantined. The post-control samples for
intestinal spirochaetes were taken at 5- to 14-month intervals, with the last samples collected in February 2002.
29
5 Results
5.1 Subspecies analysis of Brachyspira isolates by PFGE (I , II, IV, V)
Digestion of B. pilosicoli DNA by MluI and SmaI restriction enzymes yielded 9–15 and
6–10 DNA fragments, respectively. For B. innocens strains, the corresponding figures were
12–15 and 8–10. The discriminatory power with MluI and SmaI restriction enzymes was
similar for both Brachyspira species. Problems with DNA degradation were not encountered for B. pilosicoli. A distinct macrorestriction profile (MRP) was not achieved for one
B. innocens strain.
In the study of B. pilosicoli isolates from 49 sow herds (I), the 131 field isolates were divided
into 54 MRPs. The 38 strains from area N were divided into 20 MRPs, and the 93 strains
from area S into 35 MRPs. From 21 herds, more than one B. pilosicoli isolate was studied;
in 14 herds only one MRP, in six herds two MRPs, and in one herd five MRPs were observed
among the isolates.
The genetic variation was slightly higher among isolates in area S (57–100%) than in area
N (67–100%). Clustering of the MRPs according to the two geographical areas was not
seen. Three MRPs in area S and one MRP in area N were shared by two herds. One MRP
crossed the geographical areas and was shared by two herds. Persistence of a single genotype was observed in herds re-sampled within three months (eradication herd) or within
three years (two herds). In one herd, where altogether five MRPs were found in two samplings spaced three years apart, one MRP from 1997 was recovered again in 2000 and had
a high resemblance to the MRP of another isolate recovered in 2000.
Genotyping of B. pilosicoli hipp- isolates by PFGE did not reveal clustering of the strains according to hippurate hydrolysis capacity. The 11 B. pilosicoli hipp- isolates and nine hippurate
positive B. pilosicoli isolates were divided into six and four MRPs, respectively. Two of the
MRPs were shared by hippurate-negative and -positive isolates obtained from same herds.
B. pilosicoli hipp- strain Br1622, which was used for the infection trial, shared an MRP with a
B. pilosicoli hipp- isolate from another herd, although these isolates had one different nucleotide position in their 16S rDNA sequences. The two B. pilosicoli hipp- isolates from necropsied
pigs in the infection trial had a common MRP with strain Br1622.
From the herd for B. pilosicoli eradication, eight B. innocens isolates were also studied by
PFGE. The five B. innocens isolates obtained after eradication had a common MRP, which
was shared by one of the two MRPs from the three isolates cultured before eradication.
5.2 16S rDNA sequences and ultrastructure
of hippurate-negative Brachyspira pilosicoli (II)
Two out of eight B. pilosicoli hipp- isolates shared the same 16S rDNA nucleotide sequence
with B. pilosicoli type strain P43/6/78T (B. pilosicoli P43). In comparison with the sequence
of P43, three B. pilosicoli hipp- isolates had ten common mismatches between nucleotide
positions 214–589. In addition, one isolate had an 11th mismatch in position 996, and
two isolates a 12th mismatch in position 1025. The database search did not reveal spiro-
30
chaete strains of phylogenetic group IV (B. pilosicoli) (Fellström and Gunnarsson, 1995) to
share any of the ten mismatching nucleotides between positions 214–589. One of the two
sequenced hippurate-positive B. pilosicoli isolates had the same ten mismatches between
positions 214–589 as the above-mentioned six hippurate-negative isolates.
In TEM images, the cell width of the two B. pilosicoli hipp- isolates was 0.27–0.28 µm, and
cell length 6.26–6.53 µm. These dimensions were fairly concordant with those of B. pilosicoli P43, the respective mean cell dimensions of which were 0.27 µm and 6.83 µm. The
cells of B. pilosicoli hipp- isolates had pointed ends and six periplasmic flagella, characters
also seen in B. pilosicoli P43.
5.3 D-ribose utilization of porcine Brachyspira spp. (III)
A preliminary unpublished study ascertained that the broth formula was feasible and prereduction of broths was not necessary for any of the porcine Brachyspira species. Six non-B.
pilosicoli and two B. pilosicoli isolates were studied for glucose utilization by the same protocol as used for the final D-ribose study (Table 2). Excluding B. hyodysenteriae reference
strain B204 (S.h. B204), all strains showed glucose utilization by a notable reduction in pH
Table 2 Difference in pH reduction units between sugar broth and sugar-free control broth
by selected Brachyspira strains using a base broth and test protocol identical to those used in the
final D-ribose utilization study
Difference in pH reduction units
between sugar broth and sugar-free control broth
Strain
Sugar
Broths pre-reduced
Broths not pre-reduced
B. innocens
ATCC type strain B256T
Glucose
1.4
1.6
B. innocens
field isolate Br2132
Glucose
1.3
1.6
B. intermedia
ATCC type stain PWS/AT
Glucose
1.3
1.7
B. murdochii
ATCC reference strain 155-20
Glucose
1.2
1.7
B. murdochii
field isolate Br2331
Glucose
1.1
1.3
B. hyodysenteriae
ATCC reference strain B204
Glucose
0.3
0.3
B. hyodysenteriae
ATCC reference strain B204
Mannose
1.0
1.5
B. pilosicoli
ATCC type strain P43
D-ribose
1.0
1.3
B. pilosicoli
field isolate Br1622
hippurate-negative biovariant
D-ribose
1.3
1.7
31
2.5
2.0
1.5
1.0
*
0.5
*
**
0.0
B. pilosicoli
(n = 60)
control
B. pilosicoli
(n = 60)
D-ribose
non-B. pilosicoli
(n = 35)
control
non-B. pilosicoli
(n = 35)
D-ribose
Figure 4 Reduction in pH units by B. pilosicoli and non-B. pilosicoli strains in TS-broth (control)
and in TSR-broth (with 1% D-ribose). The box encloses the middle 50% of results and is bisected
by the median value. The ranges are shown by the whiskers.
regardless of the pre-reduction status of the broths. Due to an unexpectedly weak glucose
utilization, S.h. B204 was tested for mannose utilization with the same formula; mannose
utilization was clearly shown.
For the three B. pilosicoli isolates tested three times, the minimum pH reduction in D-ribose
broth was 1.7, 1.6 and 1.3 units by using cell densities equivalent to 8, 4 and 2 McFarland
units, respectively. The more dense the inocula used, the lower the variation in pH reduction between consecutive tests. Thus, cell densities representing ≥ 5.0 McFarland units
were used in the final test protocol.
In the final tests for D-ribose utilization, among the 60 B. pilosicoli strains the pH reduction
by D-ribose varied from 0.95 to 2.28 units (mean 1.72), whereas in the control broth, pH
reduction varied from 0.10 to 0.49 units (mean 0.27) (Figure 4). The mean pH reduction
among the seven B. pilosicoli hipp- strains did not differ from the overall mean of the B. pilosicoli isolates. The lowest pH reduction observed in D-ribose broth did not make the results
uncertain because in such cases the pH reduction in concomitant control broth was also
below the mean. For the 35 non-B. pilosicoli strains, mean pH reduction was 0.37 units in
both D-ribose broth and control broth.
32
5.4 Experimental infection of pigs
by B. pilosicoli hipp- strain Br1622 and type strain P43 (IV)
Mean daily weight gain of the pigs after challenge did not differ between the trial groups.
Values of haematological parameters remained within the normal range, except for leucocyte count of one pig in both inoculated groups and two pigs in the control group; values
for these animals exceeded reference values in one sampling of the first week p.i. Signs
of PIS were absent during the course of the trial, other than in one pig inoculated with
B. pilosicoli P43 that had loose faeces on days 9 and 10 p.i. Brachyspira spp. or other enteropathogens were not detected in faeces samples of this pig or in any of the other trial
pigs. In colon tissue samples, however, B. pilosicoli hipp- Br1622 was re-isolated from two
pigs necropsied on days 15 and 16 p.i.
At necropsy and upon histological examination, slight inflammatory changes were observed
on the colonic and caecal mucosa for the majority of pigs in all trial groups. In silver-stained
sections, spiral-shaped bacteria were seen invading the colonic mucosa of most pigs; in
five, six and two of the pigs in groups challenged with Br1622, type strain and sterile broth,
respectively. The degree of mild colitis and typhlitis observed in HE-stained sections was
not completely concordant with observations of spiral-shaped bacteria. FISH with probes
specific for genus Leptospira and domain bacteria revealed invasive spiral-shaped bacteria
in colon and caecum samples of five, five and three pigs in the above-mentioned groups,
respectively. These FISH-positive cases only partially matched, with the cases where spiralshaped bacteria were seen with non-specific silver staining (Table 3). FISH studies with the
three probes for Brachyspira were negative.
In TEM, endoflagellated bacteria were seen invading the colonic mucosa of two pigs: in one
pig that had been inoculated with Br1622 and necropsied 17 days p.i., and in the other pig
that had been sham-inoculated and necropsied 21 days p.i. The presence of endoflagella
suggested spirochaetes. However, the number of endoflagella observed, 12–14, was not
consistent with B. pilosicoli or Leptospira spp.
L. intracellularis was not detected in the trial pigs by PCR or serology. Four of the nine sows
in the same farrowing group had antibodies to L. intracellularis in their colostrum. However, these four sows were not the dams of the pigs chosen for the trial.
33
Table 3 Pigs inoculated with B. pilosicoli hipp- strain Br1622, B.pilosicoli type strain P43 or sterile
broth (control). Pathology, histology and fluorescent in situ hybridization results are shown.
Trial
group
Inoculum
1
Br1622
1
Br1622
2
3
Necropsy
day postinoculation
Histology
Gross
pathology 1
Histopathology 2
Spiral-shaped
bacteria 3
FISH 4
7
−
+
−
+
2
9
−
(+)
+
−
Br1622
3
11
+
+
++
+
Br1622 5
4
15
++
+
++
+
Br1622 5
5
16
++
(+)
−
+
Br1622 6
6
17
+
+
++
+
Br1622
7
21
+
(+)
++
−
Br1622
8
23
++
+
−
−
P43
9
7
+
(+)
+
−
P43
10
9
−
(+)
−
+
P43
11
11
+
(+)
+
−
P43
12
15
++
(+)
++
−
P43
13
16
++
(+)
++
+
P43
14
17
+
+
+
−
P43
15
21
+
(+)
++
+
P43
16
23
++
(+)
−
−
control
17
7
−
(+)
−
−
control
18
15
+
+
−
+
control
19
17
−
(+)
++
+
control
20
23
+
+
−
−
control
21
9
+
(+)
−
−
control
22
11
−
(+)
−
−
control 6
23
21
+
(+)
++
+
Pig
1 Gross finding 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.
3 Spiral-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.
4 Fluorescent in situ hybridization. Positive (+) and negative (−) results with probes designed for domain
bacteria and genus Leptospira.
5 Br1622 re-isolated from necropsy samples.
6 Endoflagellated bacteria observed in colonic mucosa by transmission electron microscopy.
34
5.5 Eradication of B. pilosicoli (V)
During the post-control period from April 1998 to February 2002 B. pilosicoli was not detected in any sample taken from weaners or growers (Table 4). B. innocens was isolated in
each sampling, before and after eradication. Severe post-weaning diarrhoea disappeared,
with only single litters occasionally having short periods of mild diarrhoea around weaning
time. Exact data of production before and after eradication were unavailable. According to
the owner’s opinion, the daily weight gain of growers had improved markedly.
The MICs for tiamulin of three B. pilosicoli isolates obtained before eradication were ≤ 0.125
µg ml −1. The MICs for two B. innocens isolates before eradication were ≤ 0.063 µg ml −1,
whereas after eradication, the MICs for three B. innocens isolates were 0.25, 0.125 and
≤ 0.063 µg ml −1.
Table 4 Faecal samples taken from growers post-eradication, all with negative results for
B. pilosicoli
Apr. 1998
No. of samples
20
Nov. 1998 Mar. 1999 Dec. 1999 Feb. 2001
49 1,2
42 1
45 1
371
Sep. 2001 Feb. 2002
311
381
1 Primary cultures also studied by species-specific PCR.
2 Ten samples were from colons of slaughtered pigs weighing 110 kg.
35
6 Discussion
Differential diagnostics for intestinal spirochaetes was undeveloped in Finland until the
middle of the 1990s. Since the biochemical classification scheme for WBHIS was applied,
B. pilosicoli has been identified in numerous faecal samples of diarrhoeic pigs. The regional
prevalence studies conducted in the late 1990s showed high occurrence of B. pilosicoli in
herds producing rearing pigs. Farmers and field veterinarians were highly concerned about
the “new” pathogen. The subsequent ban on antimicrobial growth promoters gave a further push for investigations on PIS. There was a lack of knowledge of the epidemiology of
B. pilosicoli, control methods for PIS in Finnish pig production and laboratory diagnostics
for B. pilosicoli; the studies presented here focused on improving information in these
three areas.
6.1 Subspecies analysis of Brachyspira isolates by PFGE
PFGE has proven to be an excellent technique for studying within-species genetic variation
of Brachyspira spp. of porcine origin (Atyeo et al., 1996, 1999a; Fellström et al., 1999),
and the genetic relationship between B. pilosicoli strains from different species of animals
and humans (Atyeo et al., 1996; Rayment et al., 1997; Trott et al., 1998; Fellström et al.,
2001a) The banding patterns of macrorestricted DNA by PFGE are highly reproducible,
which enables comparison of MRPs from different studies, in and between laboratories.
The current international database libraries of genomic profiles exploit this quality of PFGE
(Swaminathan et al., 2001; Lukinmaa et al., 2004).
The DNA of all B. pilosicoli isolates of the both phenotypes could be smoothly restricted.
For a few isolates, a mixed culture of two genotypes was suggested by the very high
numbers of bands; such isolates were excluded from the study. We observed degradation
of bacterial DNA in one B. innocens isolate. A similar problem with B. hyodysenteriae has
been described by Atyeo et al. (1999a). Using HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]
ethanesulphonic acid) as a running buffer during electrophoresis instead of Tris-containing buffer has prevented DNA degradation in problematic isolates of various species of
foodborne enteric pathogens (Koort et al., 2002; Lukinmaa et al., 2004), and this technical
improvement might work with Brachyspira spp., as well.
We found the discriminatory power of PFGE to be the same when either MluI or SmaI was
used as the restriction enzyme. Due to higher number of fragments obtained with MluI,
i.e. 9–15, a number of isolates were studied solely by using this enzyme. Atyeo et al. (1996)
and Trott et al. (1998) examined 52 and 167 B. pilosicoli isolates, respectively, from various
animal species and humans by PFGE using MluI as the restriction enzyme. The number of
banding patterns obtained in these studies varied in the same range as in our study.
6.2 Molecular epidemiology of B. pilosicoli by PFGE
Nationwide molecular epidemiological studies of porcine B. pilosicoli exploiting PFGE are
few. Atyeo et al. (1996) and Rayment et al. (1997) also used SmaI or MluI as restriction
36
enzymes in their studies. They observed that porcine B. pilosicoli strains obtained from
different territories, countries and continents were genetically very diverse, and common
MRPs were found only among isolates from the same herd. B. pilosicoli strains of human,
canine, avian or porcine origin were not genetically related. Oxberry and Hampson (2003)
investigated altogether 20 B. pilosicoli isolates from two Australian pig herds by PFGE with
SmaI as a restriction enzyme. Both herds had several B. pilosicoli genotypes, one of which
was shared by the herds. The common MRP could be explained by pig trade between the
farms. Møller et al. (1999) examined Danish porcine B. pilosicoli genotypes by PFGE using
restriction enzyme BlnI. The number of isolates per herd was quite similar to ours. Altogether 26 MRPs were found among the 35 B. pilosicoli isolates from 20 Danish pig herds,
again consistent with our results, although the number of bands obtained by BlnI was not
reported by the Danish authors. They found no common MRPs between herds. This can
be explained by the lower number of herds in the Danish study, and by the volume of pork
production in Denmark being more than tenfold that in Finland.
In our study of 131 B. pilosicoli isolates from 49 sow herds, the higher genetic variation
between the isolates from area S can be explained simply by the 2.5-fold higher number
of investigated isolates obtained there. Lack of genetic clustering according to geographic
area and only five MRPs out of 54 being shared by two herds suggest that rodents, birds
and fomites do not have a major role in transmitting B. pilosicoli in Finland.
We propose a genetic recombination or mutation of B. pilosicoli strains over time based on
the close relatedness of two strains in one herd sampled three years’ apart. Recombination
among B. pilosicoli strains has been suggested on the basis of molecular epidemiological
studies of B. pilosicoli performed in villages of Papua New Guinea (Trott et al., 1998) and in
Australian piggeries (Oxberry and Hampson, 2003). The occurrence of bacteriophages has
been demonstrated among Brachyspira spp. of animal and human origin (Humphrey et al.,
1997; Calderaro et al., 1998; Stanton et al., 2003). VHS-1, the prophage of B. hyodysenteriae, transduces genes between B. hyodysenteriae strains (Stanton et al., 2001). Transduction of genes may occur among other Brachyspira species, as well (Stanton et al., 2005).
Clustering analysis of the Finnish B. pilosicoli genotypes indicated that occurrence of B.
pilosicoli in Finnish sow herds is endemic, and replacement animals are a likely source
of new B. pilosicoli genotypes in a herd. The low mean density of pig farms (ca. one per
40 km2 in pork production areas), the Finnish climate, the rarity of outdoor housing systems and the hygiene control measures adopted diminish the vector’s role in transmitting
infection between herds.
In our work and in the above-mentioned Danish study, the MRPs obtained from nondiarrhoeic herds did not form clusters of their own. Concomitant enteric pathogens were
not recorded. However, the MRPs obtained by PFGE probably do not possess characters
predictive of the degree of pathogenicity among B. pilosicoli strains.
The MRPs of 11 hippurate-negative and nine hippurate-positive Finnish B. pilosicoli isolates
in Study II did not form clusters of their own; a single MRP could include both hippurate-negative and hippurate-positive isolates. Furthermore, certain B. pilosicoli hipp- strains
shared a common MRP despite differences in their 16S rDNA nucleotide sequences. Thus,
PFGE does not discriminate between hippurate-negative and -positive B. pilosicoli strains.
37
6.3 Characterization of hippurate-negative Brachyspira pilosicoli
Whether a single bichemical aberration of a Brachyspira species is related to the 16S rDNA
nucleotide sequence has not been widely studied. Fellström and colleagues (1999) examined rare indole-negative B. hyodysenteriae isolates of Belgian and German origin. 16S
rDNA sequences from six of these strains were identical, showing a unique nucleotide position not reported in any other Brachyspira strain. However, the uniform MRPs in their PFGE
study of these isolates suggested a common source for these isolates, and as such, the
unique 16S rDNA sequence could not be linked to indole-negativity of B. hyodysenteriae.
We showed that hippurate-negativity of B. pilosicoli is not related to any phylotype achieved
by PFGE or 16S rDNA sequencing. We noted that hippurate-negative and -positive isolates
can share a 16S rDNA sequence and possess the same MRP. These kinds of B. pilosicoli
pairs were isolated from the same herds, strengthening the assumption of a common
clone. Presumably, B. pilosicoli can downregulate the expression of hippurate hydrolase for
some unknown reason. In our earlier studies, lack of expression for hippurate hydrolysis
was irreversible in vitro. Campylobacter jejuni (C. jejuni) is the only Campylobacter species
with hippurate hydrolysis capacity, but all C. jejuni strains do not express hippurate hydrolysis despite the presence of the hippuricase gene (Chan et al., 2000). A specific PCR targeting the hippuricase gene of C. jejuni has been shown to be a useful tool for differential
diagnostics of Campylobacters (Hani and Chan, 1995; Linton et al., 1997). B. pilosicoli, as
well as an avian spirochaete, B. alvinipulli, are the only characterized Brachyspira species
with hippurate hydrolysis capacity. The genetic basis of their hippurate hydrolysis has not
been studied. The diagnostic methods targeting the hippuricase gene for these Brachyspira
species might be worth of exploring, particularly in view of the discrepancies observed in
the current PCR applications for B. pilosicoli (Thomson et al., 2001; Jansson et al., 2005)
and the lack of a species-specific PCR for B. alvinipulli.
The 16S rDNA sequences from both hippurate-negative and -positive B. pilosicoli isolates
revealed strains possessing up to 12 nucleotide mismatches compared with the sequence
from type strain P43. Species in the genus Brachyspira are phylogenetically more closely
related than species in the other genera in the class Spirochaetes (Paster et al., 1991).
Similarity in 16S rDNA nucleotide sequences within the genus Brachyspira and of strains of
B. pilosicoli isolated from pigs has been reported as 98.1–99.6%, and 99.9%, respectively
(Pettersson et al., 1996). In our study, the lowest similarity between B. pilosicoli isolates
was 98.6% indicating that the genetic diversity among B. pilosicoli strains is higher than
reported previously.
In TEM, the cell dimensions and other characteristics were similar between B. pilosicoli hippand type strain P43. The ultrastructural study was justified because of the chance of uncovering new intestinal spirochaetes in pigs. For example, wild birds have been shown
to carry spirochaetes that mimic B. pilosicoli in routine PCR (Jansson et al., 2005), and
transmission of intestinal spirochaetes between wild birds and pigs has been suggested
(Råsbäck et al., 2005).
38
6.4 D-ribose test in differentiation of porcine Brachyspira spp.
The substrate consumption of a bacteria can be detected indirectly by comparing the
growth rates in two broths where the requirements for minimum growth are fulfilled and
the substrate under investigation is present in one broth. This method has been applied
when several Brachyspira species have been characterized in detail (Trott et al., 1996c;
Stanton et al., 1997, 1998). We applied the method tentatively for several Brachyspira isolates, but in daily use it is arbitrary and liable to failure (Mäkelä, 2005; unpublished data).
Utilization of a single sugar can also be detected by measuring the reduction in pH of a
broth culture (Özcan and Miles, 1999) or a culture on solid agar (Phillips, 1976). In our
laboratory, we found the method based on pH change in broth culture promising for detection of carbohydrate utilization in Brachyspira spp.; when B. pilosicoli inoculum in a test
broth was dense, the reduction in pH was significant and rapid in the presence of D-ribose
(Mäkelä, 2005). The pH reduction was not hampered by the sphere transformation of the
spirochaetes, which often emerged at the end of the incubation period. Sphere formation leads to decreased turbidity in the broth, thus invalidating the detection of enhanced
growth by optical methods.
Carbohydrate fermentation of Brachyspira bacteria can also be determined by using solid
media (Phillips, 1976; Lemcke and Burrows, 1981). The growth of Brachyspira on solid agar
media is, however, relatively slow, whereas our method yields a result within two days.
In addition, the agar plates expire sooner than sealed broths when stored, an important
consideration in routine use.
In our study, FCS supplement in the broth caused slightly higher background on pH reduction with non-B. pilosicoli isolates than with B. pilosicoli, as seen in the results of sugarfree control broths (Figure 4). This somewhat contradicts the observations of Trott et al.
(1996), who observed higher background growth with B. pilosicoli type strain than with
B. hyodysenteriae and B. innocens type strains in sugar-free heart infusion broth with 7%
serum. Our method differed from that of Trott et al., and thus, other factors in addition
to the undefined compounds in serum might cause background growth of spirochaetes in
sugar-free media.
The 60 B. pilosicoli and 35 non-B. pilosicoli isolates and strains in this study were chosen
to represent diverse clones as far as possible. According to the results, only B. pilosicoli
among porcine intestinal spirochaetes used D-ribose. Because only three of the isolates
originated from other species of animals, any conclusion must be limited to porcine intestinal spirochaetes.
B. pilosicoli has been isolated yearly in Finland from an average of 25 herds, and B. pilosicoli hipp- from 2–7 herds. In this work, seven out of 60 B. pilosicoli isolates were of the
hippurate-negative phenotype, which justifies the conclusion that D-ribose utilization is a
more constant trait of B. pilosicoli than expression of hippurate reaction. The D-ribose test,
based on recording of pH reduction, seemed to be fairly flexible with regard to culture conditions and timing. Consequently, the D-ribose test in routine practice would strengthen
the current diagnostic scheme for porcine Brachyspira spp. infections (Table 5).
39
Table 5 Reinforced scheme for phenotypic differentiation of porcine weakly β-haemolytic
intestinal spirochaetes
Species
Indole
production
Hippurate
hydrolysis
α-galactosidase β-glucosidase
activity
activity
D-ribose
utilization
B. pilosicoli
−
±
±
−
+
B. intermedia
+
−
−
+
−
B. murdochii
−
−
−
+
−
B. innocens
−
−
+
+
−
Applicability of carbohydrate utilization tests for identification of other Brachyspira species
in the WBHIS group should be investigated. Trott et al. (1996d) and Stanton et al. (1997)
showed B. intermedia type strain PWS/AT to be negative for mannose utilization, differentiating it from type strains of B. pilosicoli, B. innocens and B. murdochii. B. intermedia is the
only species in the WBHIS group with a positive indole reaction. However, Fellström et al.
(2001a) found one indole-positive B. pilosicoli of canine origin. Furthermore, our unpublished studies of intestinal spirochaetes from poultry revealed indole-negative strains that
were positive by B. intermedia-specific PCR, and vice versa, indole-positive WBHIS strains
with negative PCR results for B. intermedia. Since B. intermedia is a poultry pathogen, and
also isolated in pigs, further efforts should be directed at improving its identification.
6.5 Aspects of experimental infection of pigs
with B. pilosicoli strains
Our preliminary hypothesis was that experimental infection with B. pilosicoli type strain
P43 would cause at least mild typhlocolitis and softened faeces in pigs with a positive reisolation of P43. For B. pilosicoli hipp- Br1622, our expectations were similar; our results from
genetic and morphological investigations of hippurate-negative isolates, and observations
of atypical Brachyspira spp. isolates elsewhere (Thomson et al. 1998, Thomson et al. 2003),
were not suggestive of decreased or lack of pathogenicity of Br1622. The design of our
study had features that might explain the silence or absence of infection. Attenuation of
the inocula strains must also be considered. Failure with the technical challenge procedure
or pre-challenge death of the inocula was very unlikely. The volume and frequency of inocula should have been adequate since an inoculation of 109 cells ml −1 of B. pilosicoli has
been satisfactory for induction of PIS when given once (Thomson et al., 1997, 2001), twice
(Neef et al., 1994) or thrice (Jensen et al., 2004).
When actively growing bacteria are inoculated in an acid stomach as a broth culture, the
number of viable cells entering the hind gut might not reach the threshold of an infective
dose. In theory, a bacteria mass scraped from a solid agar culture for immediate inoculation might form cell clumps that protect some of the bacteria. At the active growth state
the bacteria are most vulnerable to external stress. We can speculate that dormancy of
spirochaetes in the sphere stage might be advantageous for successful peroral infection.
In our experience, the comma and sphere formation of Brachyspira bacteria emerges fast
40
when the culture has consumed the nutrients of the media and when the broth culture is
left unagitated in a cool place. Our meticulous nursing of the challenge cultures, including
strict heat control of bacteria containers until inoculation, may have turned against the
success of infection.
We re-isolated only B. pilosicoli Br1622, and merely from two pigs at necropsy. For subclinical infection, the sensitivity of the culture method might be inadequate. For B. pilosicoli, the culture sensitivity is approximated as 5.4 × 105 CFU g −1 (Stege et al., 2000) or
1.5 × 102 CFU g −1 in faeces (Fellström et al., 1997), and according to our unpublished
studies, the sensitivity lies between these figures. Jensen (2005) has shown FISH to be
a sensitive method in general, especially when tissue samples have been taken prior to
post-mortem autolysis. When he compared the sensitivity of FISH and a culture method for
B. hyodysenteriae and B. pilosicoli, FISH proved to be superior for B. hyodysenteriae, but
for B. pilosicoli, the culture method was better. In our study, FISH did not reveal Brachyspira
bacteria, not even from the two pigs with a positive re-isolation of Br1622. Possibly, neither
the isolation method nor FISH can detect B. pilosicoli when the infection is subclinical.
The strain for the B. pilosicoli type strain P43 had first been reported in 1980 by Taylor et
al., being deposited in the American Type Culture Collection far later by Trott et al. (1996c).
The number of passages before its final deposition is unknown; however, some attenuation of the type strain is possible. The Finnish strain Br1622 had undergone 4–6 passages
before its deposition at −70°C, and a further four passages for the final inocula. It is unlikely that the Finnish strain was more attenuated than the type strain.
L. intracellularis-free experimental pigs were achieved by the early transfer of piglets, which
was probably aided by the low infection pressure in the herd of origin. B. pilosicoli together
with particularly L. intracellularis seem to cause aggravated growers’ diarrhoea in the field
(Thomson et al., 1998; Jacobson et al., 2003). In our study, temperature variation and
draught were minimal in the barrier rooms, and pen space per pig increased gradually
due to the necropsy schedule. The favourable environmental conditions and freedom from
concomitant pathogens could have promoted the health of the experimental pigs.
The feed of the experimental pigs was granulated without heating. Heat pelleting of feed,
which might alter the sites of digestion for certain nutrients, has been shown to increase
the prevalence of WBHIS and non-specific colitis in pig herds (Smith and Nelson, 1987;
Stege et al., 2001). Clinical SD is prevented by experimental feed with no insoluble carbohydrates (Pluske et al., 1998). According to Hampson et al. (2000), a similar experimental
feed did not prevent PIS but did reduce shedding of B. pilosicoli. However, a feed mainly
digested in the fore gut might have some controlling impact on B. pilosicoli infection.
In our experience, the histological sections from the large intestine of non-diarrhoeic weaners and growers frequently show mild inflammatory lesions, while no enteropathogenic
microorganism is detected. The mild mucosal inflammation might be solely a response to
the abundant, high-energy feed needed to meet the growth requirements of the modern
pig. In our study, mild inflammatory changes were seen microscopically on colonic mucosa
of each experimental pig, including the controls, but the degree did not clearly correlate with the observation of spiral-shaped bacteria. Thus, the slight mucosal inflammation
could be non-specific in nature.
41
Separation of the piglets from the sow into a barrier at the age of 11 days did not prevent transmission of certain enteroinvasive spiral-shaped bacteria. The identity of the endoflagellated bacteria visualized by TEM remained unknown, but based on their high number
of endoflagella, B. pilosicoli and the genus Leptospira could be excluded. If the invasive
spirochaetes visualized by FISH using the Leptospira probe were true Leptospira, it would
be concordant with the observation that suckling piglets can become infected with Leptospira via sow’s milk (Ellis, 1999).
The true virulence of B. pilosicoli hipp- Br1622 strain remained uncertain in our infection
trial. Further infection studies with B. pilosicoli hipp- should be done with pigs raised in less
favourable conditions than those here, and using a definitely unattenuated, hippuratepositive B. pilosicoli field isolate as a positive control.
6.6 Eradication of B. pilosicoli
Our attempt to eradicate B. pilosicoli from a 60-sow farrowing herd was successful. Due
to the shelters available in the vicinity of the premises, none of the growing pigs had to
be sold untimely, reducing costs to the farmer. All improvement in pigs’ health can not be
attributed solely to the disappearance of B. pilosicoli. Renovation measures undertaken on
the premises improved the general hygiene on the pig farm, undoubtedly promoting the
good health of the animals.
In Norway, one eradication attempt for L. intracellularis and B. pilosicoli was carried out in
a small 35-sow farrow-to-finish unit in summer 1998 by methods comparable with ours
(Flø et al., 2000). During the 20-month follow-up B. pilosicoli and L. intracellularis were not
detected and the clinical diarrhoea among growers had disappeared. In our study, the presence of L. intracellularis before eradication was not examined. While the dosage of 200
p.p.m. tiamulin should be adequate to control clinical symptoms and prevent pathological
lesions caused by L. intracellularis (McOrist et al., 1996), it is unclear whether this dose for
18–30 days is sufficient for its eradication. However, one can speculate that in fact L. intracellularis was the organism that had been eradicated, and the improved health was due to
the disappearance of co-factors for intestinal spirochaetosis.
L. intracellularis survives in a terrestrial environment up to two weeks (McOrist, 1997;
Collins et al., 2000) and is transmitted from sows to piglets rapidly (McOrist and Gebhart,
1999; Møller, 1999). The permanent eradication of L. intracellularis from a pig herd is considered to be very difficult (Waddilove, 1997). The risk of reinfection by fomites, rodents
or avian carriers is high (Collins et al., 1999), and if reinfection occurs, an acute and more
severe form of Lawsonia-induced disease, proliferative haemorrhagic enteritis (PHE), is to
be expected (McOrist and Gebhart, 1999). Our eradicated herd was studied for L. intracellularis for the first time in September 2001 and again in May 2002. In both samplings,
L. intracellularis was detected by nested-PCR in the faeces of weaners with mild diarrhoea.
However, after eradication measures, no period of PHE-like severe diarrhoea had been observed. L. intracellularis had probably survived in the sows or returned to sows soon after
the medication period of the eradication program. This assumption would strengthen the
conclusion that B. pilosicoli had been an essential cause of health problems in the herd.
42
At least one B. innocens genotype persisted in the herd through eradication. A similar observation was made in Norway, where Flø et al. (2000) studied eradication of Brachyspira
spp. and L. intracellularis from two small sow herds; B. innocens in the first herd and B.
murdochii in the second herd were isolated from samples before and after the eradication
program. Reinfection by B. innocens or B. murdochii from the environment can not be excluded, although the host range of these species and their survival in the environment has
not been widely investigated. Trott et al. (1996a) studied the genetic relateness amongst
Brachyspira spp. isolates from pigs, rats and several avian species by using MLEE. They observed B. murdochii isolates from rats and pigs to cluster together. We have recently found
Brachyspira spp. in abundance in Finnish free-range and indoor-housed laying hens as well
as in captive pheasants, and a portion of the isolates have been positive by PCR targeting
nox gene region specific for both B. innocens and B. murdochii (unpublished data). Thus,
cross-species transmission of B. innocens and/or B. murdochii may be possible, and the B.
innocens clone in our eradication study might have been silent in environmental animals.
Because the number of B. pilosicoli eradications attempted is low, determination of the
success rate is unreliable. Today, the mean number of sows in herds in intensive pork production areas is higher than in the trial herds presented. A large herd size and the limited
facilities available might pose a risk for failure of eradication. On the other hand, the cold
climate and the low population density in Finland and Norway indirectly assist in the eradication of B. pilosicoli.
The selection of antimicrobial agents approved for treating pigs is limited in Finland; only
tylosine, lincomycin or tiamulin can be used to treat enteric spirochaetoses (MAF, 2003).
All Finnish B. hyodysenteriae isolates have been resistant to tylosine since 1997, and their
resistance to lincomycin has increased to approximately 90%. All B. pilosicoli isolates have
been resistant to tylosine since 1998 (MAF, 2000; EELA’s unpublished data). In recent years,
some B. pilosicoli and B. hyodysenteriae strains with intermediate sensitivity to tiamulin
(MIC 1.0–2.0 µg ml −1) have been observed in EELA (unpublished data). The emergence of
multiresistant B. pilosicoli strains due to continuous usage of macrolides and pleuromutilins
on certain pig farms is a significant concern.
The national herd health program for multipliers does not necessitate freedom from B.
pilosicoli. Thus, B. pilosicoli-free herds have problems in purchasing clean replacement
animals. Regular medication of purchased pigs in quarantine is an unsustainable strategy
because of the threat of antimicrobial resistance. A nationwide eradication program for B.
pilosicoli in the near future is not likely. However, preparation for the emergence of multiresistant Brachyspira spp. should include encouraging the eradication of B. pilosicoli.
43
7 Conclusions
1 Porcine B. pilosicoli strains are genetically very diverse in Finland, and the genotypes
obtained by PFGE do not cluster according to the major pork production areas. A single B.
pilosicoli genotype can persist in a herd for several years.
2 Finnish B. pilosicoli strains possess from zero to 12 different nucleotide positions in the
partial sequence of their 16S ribosomal DNA compared with the B. pilosicoli type strain.
This within-species genetic variation (≥ 98.6%) is higher than that reported previously.
3 Hippurate-negative and -positive B. pilosicoli strains are phylogenetically indistinguishable according to PFGE and 16S rDNA sequence analysis. B. pilosicoli may downregulate
the expression of the hippurate hydrolase, and consequently, the hippurate test is inadequate for differentiation of all B. pilosicoli isolates.
4 D-ribose utilization differentiates porcine B. pilosicoli from the other porcine Brachyspira species. Supplementation of the differential diagnostic scheme with the D-ribose test
strengthens the phenotype-based diagnostics of porcine B. pilosicoli.
5 Hippurate-negative B. pilosicoli can colonize the large intestine of a pig. Further research
is needed to clarify its true pathogenicity.
6 Chronic B. pilosicoli infection can be eradicated from a 60-sow farrowing herd without
total depopulation and freedom from this infection maintained for at least four and a half
years. Low MIC value of the B. pilosicoli strain to the antimicrobial compound used and
medication of replacement animals in quarantine are apparent preconditions for successful
eradication.
44
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