Reference Work Entry

The Prokaryotes

pp 330-356

The Genus Brachyspira

  • Thaddeus B. Stanton

Phylogeny and Taxonomy

Brachyspira is the sole genus assigned to the proposed Family “Brachyspiraceae” in the Order Spirochaetales (Paster and Dewhirst, 2000). The Order Spirochaetales contains all spirochetes. The spirochetes represent a monophyletic lineage and a major early branch in eubacterial evolution (Paster et al., 1984; Paster and Dewhirst, 2000). Brachyspira cells share with other spirochetes several properties that distinguish them from other bacteria. These include a helical cell shape (Fig. 1) and a cell ultrastructure that features internal periplasmic flagella, 16S rDNA sequences with spirochete signature nucleotide bases, and a natural resistance to the antibiotic rifampin (Paster and Dewhirst, 1997; Paster and Dewhirst, 2000). Brachyspira species are readily differentiated from other spirochete genera based on comparisons of their 16S rDNA sequences (Paster et al., 1991; Paster and Dewhirst, 1997).

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Fig. 1.

A) Brachyspira alvinipulli C1T cells and B) B. hyodysenteriae B78T cells. Phase contrast photomicrographs of wet mount preparations. Marker bars = 10 µm. From Stanton et al. (1998), with permission.

General Characteristics

Brachyspira species have been isolated from animal or human intestinal contents, feces-contaminated habitats (e.g., duck ponds), and human blood. They are anaerobic bacteria but are aerotolerant due, at least in part, to high NADH oxidase activity. They use soluble sugars as carbon and energy sources. Growing cells of Brachyspira species (B. aalborgi has not been investigated) consume low concentrations of oxygen via NADH oxidase and produce acetate, butyrate, H2 and CO2 from glucose. Some species also produce ethanol. Brachyspira DNAs have a low G+C content (24.5–26.7 mol%; undetermined for B. aalborgi). Brachyspira species have high 16S rDNA sequence similarities with each other. Brachyspira strains have been assigned to eight electrophoretic type (ET) groups based on multilocus enzyme electrophoresis (MEE) analysis. The MEE groups correspond to seven species as defined from DNA homology studies and one currently uncharacterized provisional species (“B. canis”). Uncharacterized intestinal spirochetes of chickens are likely to represent a new Brachyspira species, “B. pulli” (Gabe et al., 1998; Stephens and Hampson, 2001). It has been recommended that uncharacterized/uncultured spirochetes from the human intestine be named “B. christiani” (Jensen et al., 2001). 1

1

Brachyspira Species

There are seven recognized species of the genus Brachyspira (Table 1). The species B. hyodysenteriae, B. pilosicoli, B. alvinipulli and B. intermedia have been shown to cause disease when inoculated as pure cultures into their healthy, natural hosts. The type strain of each species is available from the American Type Culture Collection (ATCC; Table 1) and these strains should be included in studies of Brachyspira species.

Table 1.

Brachyspira species, pathogenicity, and animal hosts.

Species

Type strain

ATCC number

Intestinal origin

Demonstrated pathogenicity (animal)a

B. hyodysenteriae

B78T

27164

Swine and rheas

Yes (swine)

B. innocens

B256T

29796

Swine

No

B. intermedia

PWS/AT

51140

Swine and chickens

Yes (chickens)

B. pilosicoli

P43/6/78T

51139

Swine, birds, dogs, humans, and nonhuman primates

Yes (swine)

   

Yes (chickens)

B. murdochii

56-150T

51254

Swine and rats

No

B. aalborgi

513AT

43994

Humans

No

B. alvinipulli

C1T

51933

Chickens

Yes (chickens)

aPure cultures of either the type strain or other strains cause disease when inoculated into normal, healthy host animals.

History of Brachyspira Taxonomy

In the past 25 years, there have been several taxonomic changes for spirochetes now assigned to the genus Brachyspira. Initially, the designation “Treponema hyodysenteriae” was applied to both pathogenic (strongly hemolytic) and nonpathogenic (weakly hemolytic) strains of intestinal spirochetes from swine. By analysis of DNA-DNA relative reassociation, Miao and colleagues found that these pathogenic and nonpathogenic strains share only 28% sequence homology (Miao et al., 1978). This low sequence homology led to the reclassification of the nonpathogenic strains as a new species, Treponema innocens (Kinyon and Harris, 1979). In 1991, Stanton and colleagues proposed a reclassification of T. hyodysenteriae and T. innocens to a new genus “Serpula” based on 16S rRNA sequence analysis, DNA-DNA relative reassociation (S1 nuclease method), protein electrophoretic profiles, and genomic DNA restriction endonuclease analysis (Stanton et al., 1991). The genus name Serpula was then changed to “Serpulina” after it was determined that Serpula had prior use as a name for a genus of fungi (Stanton, 1992). Most recently, Ochiai et al. (1997) proposed the unification of the genera Serpulina and Brachyspira. The genus name Brachyspira was first given to a human intestinal isolate, Brachypsira aalborgi, in 1982 (Hovind-Hougen et al., 1982). Owing to the use of the genus name Brachyspira prior to the genus designation Serpulina, this proposed change is consistent with international taxonomic rules governing bacterial nomenclature and should be followed. Unfortunately, at the same time, the action has raised Brachyspira aalborgi to the status of type species of the genus Brachyspira. Unlike strains of the other Brachyspira species (especially hyodysenteriae and pilosicoli), only strain 513AT of B. aalborgi has undergone a very limited characterization (Hovind-Hougen et al., 1982; Ochiai et al., 1997). More extensive biochemical, physiological and genetic investigations of this species are clearly needed. Brachyspira aalborgi is currently considered a commensal species of the human intestinal microbiota. Finally, in older literature references, the species B. pilosicoli was given the provisional designation “Anguillina coli” (Lee et al., 1993b; Park et al., 1995).

Multilocus Enzyme Electrophoresis (MEE) Analysis

MEE analysis is a useful molecular typing technique for differentiating, identifying and determining phylogenetic relationships of taxonomically related bacteria and for determining their phylogenetic relationships (Selander et al., 1986). Based on MEE analysis of 15 constitutive enzymes, Hampson and colleagues (Lee et al., 1993a; Lee et al., 1993b; Lee et al., 1993c; Lee and Hampson, 1994; Swayne et al., 1995; Trott et al., 1996a) have assembled hundreds of intestinal Brachyspira strains into MEE groups (Stanton et al., 1996; Duhamel et al., 1998). The DNA homologies between strains in different MEE groups range from 21 to 64% and the homologies between strains within the same MEE group are 78% or greater (Stanton et al., 1991; Stanton et al., 1997b; Stanton et al., 1998; Trott et al., 1996c). The recommended phylogenetic definition of a species includes strains with 70% or greater DNA-DNA relatedness and with 5°C or less ΔTm (Wayne et al., 1987). Thus, the MEE technique can be used for the presumptive identification of Brachyspira species and for predicting new species. The single best “stand alone” technique for confirmed identification of a new Brachyspira species is DNA-DNA relative reassociation using the S1 nuclease method (Crosa et al., 1973; Grimont et al., 1980).

16S rDNA Sequence Analyses

Comparative analysis of 16S rDNA (rrs gene) sequences has played an essential role in establishing a phylogeny-based classification of the spirochetes (Paster and Dewhirst, 2000). Based on 16S rDNA sequence comparisons, the Brachyspira represent a distinct line in spirochete evolution (Paster et al., 1991; Stanton et al., 1991; Paster and Dewhirst, 1997). Consequently, 16S rDNA sequence determinations are important for classifying these enteric bacteria as spirochetes and for identifying them as members of the genus Brachyspira (Olsen et al., 2000).

Partial rrs sequences have been obtained for intestinal spirochetes from various animal hosts and humans (Hookey et al., 1994; Fellstrom et al., 1995; Pettersson et al., 1996; Stanton et al., 1996; De Smet et al., 1998; Kraaz et al., 2000). Intestinal spirochete dendrograms based on 16S rDNA sequence comparisons correlate with the genetic groupings based on MEE analysis (Stanton et al., 1996).

In view of the substantial rrs gene similarity among Brachyspira species (Table 2), it is recommended that new species be designated only after their phylogenetic relationships with known Brachyspira species are confirmed by additional techniques (Stanton et al., 1996). One additional method is MEE analysis (MEE analysis). Another method, the conventional “gold standard” technique for identification of bacterial species, is DNA sequence homology estimation (Wayne et al., 1987). For estimating DNA sequence homology, DNA-DNA relative reassociation using the S1 nuclease method with ΔTm evaluation is recommended (Crosa et al., 1973; Grimont et al., 1980).

Table 2.

Brachyspira species characteristics.

Species

Hemolysis type

Growth ratea

Cell size (µm)

Flagella per cell

Number of ETs

DNA G+C

16S rDNA signatureb

16S rDNA sequence similarityc

Indole production

Hippurete hydrolysis

B. aal. 513AT

B. hyo B78T

B. aalborgi d

Weak

NR 7–14 d

2–6 × 0.2

8

N/A

NR

No

100

96.5

+

B. hyodysenteriae

Strong

3–5 h 3–4 d

7–9 × 0.3–0.4

22–28

29

25.9

No

96.5

100

+/−

 

B. intermedia

Weak

2–4 h 3–5 d

8–10 × 0.35–0.45

24–28

7

25

No

96.0

99.1

+

+

B. innocens

Weak

3–5 h 3–5 d

7–9 × 0.3–0.4

20–26

10

26

No

96.5

99.5

 

B. pilosicoli

Weak

1–2 h 3–4 d

5–7 × 0.23–0.3

8–12

33

24.6

Yes

96.4

98.5

+/−

+/(−)

B. murdochii

Weak

2–4 h 3–5

5–8 × 0.3–0.4

22–26

10

27

No

95.9

98.5

B. alvinipulli d

Weak

3–5 h 3–5 d

8–11 × 0.2–0.35

22–30

N/A

24.6

No

95.7

98.4

+

Symbols and abbreviations: ET, electrophoretic types; +, positive; −, negative; (−), indole not produced or hippurate not hydrolyzed; NR, not reported; and N/A, not applicable.

aPopulation doubling time in culture broth and days for colony development on agar-containing media.

bA unique signature sequence of nucleotides was identified within the 16S rDNA of B. pilosicoli and has been used to design specific polymerase chain reaction (PCR) tests for this species (Park et al., 1995; Fellstrom et al., 1997).

c16S rDNA values based on type strains except for B. murdochii 155–20. Based on GenBank sequences: Z22781, U14930, U23033, U14920, U14927, U22838, and U23030.

dOnly type strains 513AT and C1T of B. aalborgi and B. alvinipulli, respectively, have been extensively characterized.

Information from Harris et al. (1972); Hovind-Hougen et al. (1982); Kinyon and Harris (1979); Stanton et al. (Stanton et al., 1991; Stanton et al., 1996; Stanton et al., 1997b; Stanton et al., 1998); Fellstrom et al. (Fellstrom et al., 1995; Fellstrom et al., 1997; Fellstrom et al., 1999); McLaren et al. (1997); Trott et al. (Trott et al., 1996a; Trott et al., 1996b; Trott et al., 1997, Trott et al., 1997, Trott et al., 1997); DeSmet et al. (1998); Duhamel et al. (1998); and Kraaz et al. (2000).

16S rDNA of Uncultured B. aalborgi-like Species

Recent studies suggest there are uncharacterized Brachyspira species phylogenetically more closely related to B. aalborgi than to B. hyodysenteriae (Pettersson et al., 2000). Rrs genes were amplified from unknown bacteria within colonic biopsy samples of two human patients. Seventeen spirochetal-like sequences resembled those of B. aalborgi. Three clusters of sequences could be differentiated. One contained the two cultivated strains of B. aalborgi. Although only 0.5–1.3% of the 16S rDNA sequence is different between clusters, this same level of sequence difference exists between different Brachyspira species (Table 2). Thus, the strains more than likely represent one or two new Brachyspira species (Pettersson et al., 2000).

By contrast to these studies analyzing almost complete 16S rRNA sequences of intestinal spirochetes, other investigators have relied on sequences of short sections (i.e. 433 bp and 241bp) of 16S rRNA genes for the identification of Brachyspira aalborgi strains (Koteish et al., 2003; Munshi et al., 2003). The highly conserved nature of the16S rRNA sequences of known Brachyspira species and the potential diversity of human intestinal spirochetes make these identifications, at best, presumptive. Important in determining the significance of intestinal spirochetes to human health will be the characterization of the HIS-specific spirochetes in pure culture.

23S rDNA Sequence Analysis

Approximately 2,470-bp of the 23S rDNA genes of B. hyodysenteriae, B. innocens, B. intermedia, B. murdochii and B. pilosicoli strains have been sequenced (Leser et al., 1997). The 23S rDNA sequences are highly (96.8–99.8%) similar. A phylogenetic tree based on sequence comparisons is consistent with 16S rDNA-based and MEE-based comparisons of these species. Various Brachyspira species can be differentiated by 23S rDNA-targeted polymerase chain reaction (PCR) assays (Leser et al., 1997) or by PCR-restriction fragment length polymorphism (RFLP) analysis of 23S rRNA genes (Barcellos et al., 2000).

Habitat

Brachyspira species colonize the lower intestinal tracts (ceca and colons) of animals and humans. Brachyspira pilosicoli, B. aalborgi, B. hyodysenteriae and B. alvinipulli have been observed in close physical proximity to epithelial tissues lining the intestinal tract. Intestinal mucus secreted by goblet cells is likely to be important both as a physical matrix and as chemical substrate for these spirochetes in their microhabitats. Various cell traits are considered or have been demonstrated to be important for Brachyspira to colonize the intestinal tract (see Ecology; Disease).

Cells of B. pilosicoli, a species isolated from various animal hosts, and B. aalborgi, a species so far isolated only from humans, can colonize intestinal mucosal surfaces by attaching to enterocytes (Barrett, 1997; Swayne and McLaren, 1997; Taylor and Trott, 1997; Mikosza et al., 1999; Jensen et al., 2000). One end of the spirochete cell attaches to an epithelial cell and the other end extends away from the surface of the epithelial cell (Fig. 2). Densely packed, parallel arrays of spirochetes in close proximity form on the epithelial surface in this way. The density of spirochetes in these assemblages is estimated to be 20–80 spirochete cells per enterocyte (Stanton, 1997a). Although end-on attachment is a characteristic feature of B. aalborgi and B. pilosicoli colonization, it may not be a sine qua non for colonization and disease (Thomson et al., 1997; Jensen et al., 2000).

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Fig. 2.

Scanning electron micrograph of the colonic mucosa of a pig with colitis showing the typical appearance of intestinal spirochetosis. Epithelial cells are extensively colonized by spirochetes attached by one end to the luminal surface of the cells. Marker bar = 2 µm. From Sellwood and Bland (1997), with permission.

End-on attachment to intestinal tissues by densely packed, parallel arrays of B. pilosicoli or B. aalborgi cells creates the appearance of a “false brush border” on mucosal epithelial cells. This appearance of colonizing spirochetes has been commonly referred to as “intestinal spirochetosis” by histologists (Duhamel, 1997a; Swayne and McLaren, 1997). Intestinal spirochetosis has been observed in tissue sections both of healthy animals and humans and of individuals with intestinal disorders. Takeuchi and Zeller (1972) estimated that unidentified spirochetes attached to rhesus monkey colonic mucosa were present at a density of 1,800 bacteria·mm−2. So far, B. pilosicoli and B. aalborgi are the only characterized spirochetes associated with intestinal spirochetosis.

Attachment to intestinal tissues is not a colonization feature of B. hyodysenteriae, the agent of swine dysentery, or of B. alvinipulli, a chicken pathogen. In the early stages of swine dysentery, B. hyodysenteriae cells first colonize along the intestinal epithelium of the swine cecum and colon and then among epithelial cells and within goblet cells (Glock et al., 1974; Kubo et al., 1979; Kennedy et al., 1988). As the disease progresses, lesions appear in the mucosa at sites of spirochete colonization, and host blood passes into the intestinal lumen through the lesions (Glock et al., 1974; Kennedy and Strafuss, 1977; Kubo et al., 1979). Brachyspira alvinipulli strain C1T cells colonize chicken ceca and colons by irregular cell aggregates adjacent to intestinal epithelial cells and within villus crypts (Swayne et al., 1995; Swayne and McLaren, 1997; Stanton et al., 1998).

Microhabitats have yet to be extensively studied for B. innocens, B. intermedia, and B. murdochii.

Isolation

Physical Isolation Methods

In the first studies to identify B. hyodysenteriae as the etiologic agent of swine dysentery, the spirochete was isolated from dysenteric pigs after filtering colon contents through a 0.65 µm filter (Taylor and Alexander, 1971) and colonic epithelial tissue extracts through a series of filters of decreasing pore size (Harris et al., 1972b). The filtrates were inoculated onto agar media and incubated anaerobically to obtain isolated colonies.

Another physical isolation method takes advantage of bacterial motility to separate Brachyspira strains from nonmotile intestinal bacteria (Olson, 1996). Sterile scalpels are used to cut parallel lines in a selective agar medium containing spectinomycin (400 µg/ml). Intestinal samples are inoculated as streaks in the center of the Petri plates and perpendicular to the cut lines. The motile B. hyodysenteriae cells (strongly hemolytic) and cells of other intestinal spirochetes (weakly or nonhemolytic) migrate along the lines. The spirochetes can be isolated from agar medium samples taken at the ends of the cut lines and away from the original inoculation site.

Selective Culture Media

Brachyspira hyodysenteriae and other Brachyspira species can be isolated by inoculating intestinal contents or tissues onto solid agar culture media containing antibiotics selective for the growth of those spirochetes (Table 3). Achacha and Messier (1992) compared various selective media and found BJ medium (Table 3) with five antibiotics provided the highest rate of isolation of B. hyodysenteriae from feces of experimentally infected swine (130 of 145 samples tested). They also reported the encouraging observation that pig feces extract could be eliminated from the medium with essentially no effect on isolation success (128 of 145 samples tested).

Table 3.

Brachyspira culture media.

A) Selective isolation media

Medium

Medium components

Target species

References

TSB(S)

Trypticase-soy agar

B. hyodysenteriae

Songer et al., 1976

+ 5% bovine blood

 

Taylor et al., 1980

+ spectinomycin (400)

  

TSB(SCV)

Tryptone-soya agar

B. hyodysenteriae

Jenkinson and Winger, 1981

+ 10% sheep blood

  

+ spectinomycin (400)

  

+ colistin (25)

  

+ vancomycin (25)

  

BJ

Trypticase-soy agar

B. hyodysenteriae

Kunkle and Kinyon, 1988

+ 5% bovine blood

  

+ 5% swine faces extract

  

+ spectinomycin (400)

  

+ spiramycin (25)

  

+ rifampin (12.5)

  

+ vancomycin (6.2)

  

+ colistin (6.2)

  

TSB(SP)

Trypticase-soy agar

B. aalborgi

Kraaz, 2000

+ 10% bovine blood

 

Hovind-Hougen et al., 1982

+ spectinomycin (400)

  

+ polymyxin (5)

  

BB(SR)

Blood agar modified medium

B. hyodysenteriae

Calderaro et al., 2001

+ 7% equine blood

  

+ spectinomycin (400)

  

+ rifampin (30)

  

B)Liquid media

Medium

Medium components

References

BHIS broth (general purpose growth)

Brain-heart infusion broth (contains glucose); 10% calf serum (heat-treated 56°C, 30 min); 0.1% L-cysteine-HCl; 0.0001% resazurin

Stanton and Cornell, 1987b

HS broth (for identifying growth substrates)

Heart infusion broth; 2–10% calf serum (heart treated 56°C, 30 min); L-cysteine-HCl; 0.0001% resazurin

Stanton and Lebo, 1988

Kunkle’s broth (autoclavable medium)

Trypticase soy broth; 0.5% glucose; 0.2% NaHCO3; 0.05% L-cysteine-HCl; 1.0% yeast extract; and 2 of 3 of the following: fetal bovine serum, cholesterol, or swine facal extract

Kunkle et al., 1986

Values in table are final concentration (v/v or w/v). Antibiotic concentrations expressed as µg/ml of medium.

Brachyspira species other than B. hyodysenteriae can be isolated from swine, other animals, and humans by using selective culture media similar, if not identical, to those used for B. hyodysenteriae (Barrett, 1997; Jensen, 1997; Table 3). A note of caution is that antibiotics used to select for B. hyodysenteriae growth may select against growth of strains of certain Brachyspira species, such as B. aalborgi (Kraaz et al., 2000) and B. pilosicoli (Trott et al., 1996b). Selective culture media to directly isolate B. aalborgi from human feces have been described (Brooke et al., 2003; Calderaro et al., 2003). The media appear promising but so far have been used successfully for samples from only two patients.

Sample Handling, Inoculation, and Incubation Conditions

Brachyspira cells are aerotolerant anaerobes. Brachyspira hyodysenteriae strain B204 cells can withstand exposure to air for two or more hours (Stanton et al., 1999b). Nevertheless, whenever possible, intestinal contents or tissue samples should be protected from air exposure (for example, placed in anaerobic culture broth) and maintained on wet ice for transport to the laboratory. Swab samples of feces should be kept moist in a transport medium.

Brachyspira colonies can be isolated from intestinal samples by standard streaking methods using sterile inoculating loops to dilute bacterial cells across agar plate surfaces. Alternatively, serial tenfold dilutions of the samples can be made in tubes containing prereduced (oxygen-free) sterile basal culture broth (e.g., trypticase soy or brain heart infusion) and by spreading samples across the surface of agar plates. The latter technique, of course, enables viable colony forming units (CFU) in the original sample to be estimated.

Owing to their aerotolerant nature, preliminary steps in the isolation of known Brachyspira species (streaking agar plate, and diluting samples) can be carried out on the lab bench (in an air atmosphere). Inoculated agar plates are incubated under anaerobic conditions by placing them within an anaerobic glove box or within anaerobic Gas-Pak jars. To further reduce exposure of Brachyspira cells to oxygen, intestinal samples can be transferred into an anaerobic glove box and all subsequent processing of samples carried out within the anaerobic atmosphere.

Brachyspira pilosicoli from Blood

Brachyspira pilosicoli strains have been isolated from the blood of critically ill human patients without the use of selective agar medium (Trott et al., 1997b). Cells of the spirochete were cultured from blood samples inoculated into Hemoline blood culture medium (bioMerieux, Lyon, France), BioArgos Sanofi diagnostic anaerobic medium (Pasteur Institute, Paris, France) and ESP automated blood culture media (Becton Dickinson Difco, Detroit, Michigan, United States). A study comparing various commercial blood culture systems indicated that prolonged culture incubation (5–14 days) is necessary to detect B. pilosicoli cells (Brooke et al., 2000).

Alternative Isolation Techniques

With few exceptions, isolation techniques for Brachyspira have revolved around selective culture media containing antibiotics and developed to clinically detect B. hyodysenteriae. These methods have led to the successful isolation of seven different Brachyspira species. It seems likely that the use of alternative sources of inocula the, modification of medium components and incubation conditions, and the incorporation of physical separation techniques will yield additional Brachyspira species.

Identification of Species

Various phenotypic characteristics and PCR based tests are useful for identifying known Brachyspira species (Table 2). “Strong” hemolysis of B. hyodysenteriae colonies and a unique signature sequence in the 16S rDNA of B. pilosicoli are often used in clinical and epidemiological studies to identify those pathogens.

Colony Hemolysis

Brachyspira hyodysenteriae is the only recognized Brachyspira species that is “strongly hemolytic” (Table 2). Colonies of B. hyosysenteriae form zones of β-hemolysis on trypticase soy-blood agar plates. Diagnosis of the disease swine dysentery is based on culturing of spirochete-like bacteria forming strongly hemolytic colonies and on histological evidence of spirochete-induced lesions in the ceca and colons of dysenteric swine. Well-isolated colonies on freshly prepared blood agar plates should be examined to assess hemolysis type of an intestinal spirochete. Control cultures of Brachyspira-specific type strains should be included for hemolysis comparisons.

Cell Ultrastructure

The number of flagella per cell has been commonly used in spirochete taxonomy and identification. Brachyspira species can be divided into two groups based on flagellar numbers and cell size (Table 2). Larger size species, such as B. hyodysenteriae, B. innocens, B. intermedia, B. murdochii and B. alvinipulli, have 20–30 flagella per cell. Brachyspira pilosicoli and B. aalborgi cells are shorter in length and have 8–12 flagella per cell. As with other spirochetes, the flagella attach in roughly equal numbers at each end of the cell (Fig. 3) and wind around the spirochete cell between the protoplasmic cylinder and outer sheath, and their free ends overlap in the cell center. Accurate estimates of flagellar numbers can be obtained by transmission electron microscopy of bacteria suspended in distilled water and negatively stained (e.g., 2% phosphotungstic acid, pH 7.0). Both flagella and flagellar attachment sites at the ends of the cells can be counted (Fig. 3).

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Fig. 3.

Electron micrograph of one end of B. alvinipulli C1T cell negatively stained with 2% phosphotungstic acid (pH 7.0). Disrupted outer sheath enables insertion sites of 15 periplasmic flagella to be seen (white arrowheads). Marker bar = 0.25 µm. From Stanton et al. (1998), with permission.

Biochemical Tests

Biochemical tests for indole production (Finegold and Martin, 1982), hippurate hydrolysis (Smibert and Krieg, 1981), and commercial assay kits, such as API-ZYM (Hunter and Wood, 1979; Fellstrom et al., 1999; Stanton et al., 1998), have been used to characterize Brachyspira species (Table 2). For B. aalborgi and B. alvinipulli, only a limited number of strains have been examined.

Attempts have been made to classify porcine Brachyspira isolates based on hemolysis pattern, indole production, hippurate hydrolysis, colorimetric tests for sugar hydrolyzing enzymes, and pathogenicity for swine (Fellstrom et al., 1995; Fellstrom et al., 1997). Unfortunately, porcine strains with properties different from the expected test results have been identified (Fellstrom et al., 1997; Fellstrom et al., 1999). Additionally, the biochemical classification of Brachyspira species does not work when non-porcine strains are included (De Smet et al., 1998; Stanton et al., 1998). Many biochemical tests, such as the hydrolysis of a certain color-yielding substrate, rely on one or a few encoding genes and therefore do not provide an extensive phylogenetic basis for taxonomy.

Species-specific PCR-based Assays

PCR assays have been described for the specific detection of porcine strains of B. hyodysenteriae (Elder et al., 1994; Harel and Forget, 1995; Leser et al., 1997; Atyeo et al., 1998; Atyeo et al., 1999), B. pilosicoli (Park et al., 1995; Fellstrom et al., 1997; Leser et al., 1997; Muniappa et al., 1997; Atyeo et al., 1998), and B. intermedia (Leser et al., 1997). A restriction fragment length polymorphism (RFLP)-PCR assay targeting 16S rDNA was developed to differentiate the type strains of seven Brachyspira species (Stanton et al., 1998). Unfortunately, clinical isolates of weakly hemolytic spirochetes, Brachyspira stains, have restriction patterns that do not match those of the type strains (T. B. Stanton, unpublished observation). In addition, PCR assays targetting NADH oxidase and 16S rRNA genes have been developed to differentiate Brachyspira species and used to detect B. aalborgi and B. pilosicoli in human biopsy specimens (Atyeo et al., 1999; Mikosza et al., 1999; Mikosza et al., 2001). Also, PCR assays targeting 23S rDNA were developed to distinguish porcine strains of B. hyodysenteriae, B. intermedia (group II) and B. pilosicoli (group IV) (Leser et al., 1997). RFLP-PCR assays targeting genes encoding NADH oxidase or 23S rRNA have been developed to differentitate Brachyspira species (Barcellos et al., 2000; Rohde et al., 2002).

Investigations with additional strains of known Brachyspira species and partially characterized intestinal spirochete isolates (“B. canis”) are needed to determine whether these assays or any PCR-based assays will suffice as an individual test for identifying Brachyspira species. At this time, a likely to be reliable approach for species detection and identification would incorporate a combination of PCR-based tests for more than one gene (Mikosza et al., 1999; La et al., 2003).

Establishing New Brachyspira Species

Based on investigations of known Brachyspira species (see Phylogeny and Taxonomy), a new Brachyspira species should be characterized by 16S rDNA sequence analysis or/and MEE analysis (preferably both) of multiple isolates. Isolates (one or more strains) forming a unique 16S rDNA branch or MEE electrophoretic type should be compared by DNA sequence homology (S1 nuclease method with ΔTm estimates) with closely related Brachyspira species. Finally, the ultrastructural, cultural, and biochemical characteristics of a species help define its lifestyle and facilitate additional research. These should be determined.

Preservation and Transport of Brachyspira Cultures

Short-term Stock Cultures

Broth cultures of most Brachyspira species remain viable when stored at 5°C for short time periods of 1–2 weeks. These cultures can be used as “working” stock cultures to inoculate fresh cultures for experiments. Refrigerated cultures should be in the exponential phase of growth (for many strains, 2–4 × 108 cells/ml, direct microscope counts). Importantly, avoid exposing cultures to air. The junction between the culture tube mouth and the rubber stopper corking the tube should be firmly sealed with stretchable yellow plastic tape (3M Scotch Brand #471) before storage. These working stock cultures should be transferred every two weeks. Long-term in vitro passage of strains (with possible loss of virulence) should be avoided by starting fresh working stock cultures from long-term stock cultures every several months.

Agar plate cultures of B. hyodysenteriae can be stored at room temperature in an anaerobic atmosphere for at least a week. The plates should be sealed (Parafilm, American National Can Inc.) to prevent desiccation. A slowly growing spirochete identified as B. aalborgi remained viable when kept at room temperature in an anaerobic jar for over 3 months (Kraaz et al., 2000).

Long-term Stock Cultures

For long-term storage of Brachyspira strains, the following method is recommended. Brachyspira cultures in the exponential phase of growth in broth media (2–4 × 108 cells/ml) are harvested by centrifugation (15 min, 5,000g). The pelleted bacteria are resuspended at 50–100 times their original concentration in fresh sterile broth medium containing dimethyl sulfoxide (DMSO), 10% (v/v, final concentration). The concentrated cell suspension is dispensed into Nunc cryovials (0.5–1.0 ml/vial). The sealed vials are placed upright in a beaker containing enough 95% ethanol to equal the fluid level of the suspensions (and well below the tops of the cryovials). The ethanol bath is intended to provide a more uniform rate of freezing of the cells and, with the DMSO as a cryoprotectant, prevent ice crystals from damaging the bacteria. The beaker is placed in an ultra-cold freezer (−75°C). After 24 h, the frozen stock cultures are transferred to storage boxes in the freezer. After 7–10 d, one of the cryovials should be examined to insure recovery of contaminant-free Brachyspira cells. Brachyspira cells have remained viable in frozen stocks prepared in this way for over 15 years (T. B. Stanton, unpublished observations). Completely thawed stocks should not be re-frozen. However, it is often possible to subculture without thawing the frozen stocks, by scraping the surface of a frozen cell suspension with a sterile inoculation loop and inoculating fresh media with the ice scrapings. Brachyspira hyodysenteriae cultures also have been preserved by lyophilization (Stanton and Lebo, 1988).

Shipping Stock Cultures

Brachyspira strains can be successfully transported through the mail as frozen cell stocks on dry ice. Alternatively, agar plate cultures will survive shipment by express mail delivery, without the need for wet or dry ice. Within an anaerobic chamber, agar plates with visible colony growth are sealed shut with flexible film (Parafilm). Within the chamber, the plates are then double-sealed in two plastic bags (smaller bag within larger bag) using a heat seal device (Seal-a-Meal, Dazey Corp., Industrial Airport, KS), such as used for DNA-DNA membrane blot hybridizations, and placed in appropriate shipping containers. For overnight delivery, stoppered broth cultures can be sealed with plastic tape and mailed in special shipping containers. Appropriate government and private carrier regulations governing the packaging and shipping of bacterial cultures must be followed.

Physiology

Culture Broth Media

Various anaerobic, nutritionally complex broth media have been described for Brachyspira species (Stanton, 1997a). Brain heart infusion with serum (BHIS) broth and Kunkle’s broth are commonly used for routine growth of Brachyspira hyodysenteriae (Table 3) (Kunkle et al., 1986; Stanton and Lebo, 1988). Both media support high growth yields of this species (109 to 4 × 109 cells/ml, direct microscope counts). In BHIS broth, the population doubling times for type strains of B. hyodysenteriae, B. innocens, B. intermedia, B. murdochii and B. alvinipulli are 2–4 hours (Table 3). Brachyspira pilosicoli P43/6/78T has a doubling time of 1–2 hours. Incubation temperatures of 38–40°C (the swine body temperature is 39°C) are used for Brachyspira, and stirring of broth cultures (with magnetic stir bars) is important for optimum growth.

Heart infusion with serum (HS) broth for Brachyspira species (Table 3) requires added carbohydrates to support optimum growth yields of Brachyspira species. Consequently, HS broth is useful for identifying growth substrates (Table 4) and metabolic end products. Since animal serum contains glucose, a minimal serum concentration, i.e., one not limiting for growth, should be used in studies to identify growth substrates and products.

Table 4.

Brachyspira growth substrates.a

Compound

B. hyodysenteriae B78T

B. innocens B256T

B. intermedia PWS/AT

B. pilosicoli P43/6/78T

B. murdochii 56/150T

B. alvinipulli C-1T

None

+

+

+

+

+

+

D-Glucose

++++

++++

++++

++++

++++

++++

D-Fructose

++++

++++

++++

++++

+++

++

Sucrose

++++

+++

++++

++++

+++

NG

D-Trehalose

++++

+++

++++

++++

+++

NG

D-Galactose

++

++

++

++

NG

NG

D-Mannose

++

+++

NG

+++

++

++++

N-Acetyl-D-glucosamine

++++

++++

++++

++++

++++

++++

D-Glucosamine

+++

+++

NG

+++

NG

++++

D-Maltose

+

+++

+++

++

++

+++

D-Cellobiose

NG

+++

NG

++++

++++

NT

L-Fucose

NG

++

++

++

++

NG

Pyruvate

++

++

++

++

++

NG

D-Ribose

NG

NG

NG

++++

NG

NG

Lactose

+

NT

NG

NT

+

NG

Symbols: +, background growth (no added substrate); ++, low growth, final cell yields approximately 2- to 4-fold greater than background; +++, intermediate growth, final cell yields 4- to 10-fold greater than background; and ++++, optimum growth, final cell yields >10-fold background levels.

Abbreviations: NG, no detectable growth above background (medium without added substrate); and NT, substrate not tested.

aBased on growth studies with B. hyodysenteriae cultures in HS broth by Stanton and Lebo (1988); Trott et al. (1996), Trott et al. (1996); and Stanton et al. (Stanton et al., 1997b; Stanton et al., 1998). In those studies, additional substrates were found not to support growth of any species. Values in table refer to relative cell yields.

NT broth, a serum-free medium ultrafiltered (10,000 MW cutoff filter) to remove high MW proteins, supports B. hyodysenteriae growth (Humphrey et al., 1997). Cholesterol, an essential growth requirement for the spirochete, is added to the medium in place of serum. NT broth cultures of B. hyodysenteriae are used to purify the generalized transducing bacteriophage VSH-1 (Humphrey et al., 1997). (See Genetics.) NT broth also may be useful for identifying and purifying extracellular (protein) products of Brachyspira spp.

The oxidation-reduction potential of broth cultures may be a controlling factor for B. hyodysenteriae growth (Stanton, 1997a). The spirochete can be difficult to culture in stringently prepared anaerobic broth media unless a small amount of air is introduced into the culture oxygen-free atmosphere (Stanton and Lebo, 1988). A simple method is to inject a volume of sterile air through the rubber stopper sealing a culture vessel so that the culture atmosphere contains 1% oxygen (e.g., 1% O2:99% N2). Brachyspira hyodysenteriae cells have been successfully cultured beneath an air atmosphere in large (i.e., 12 liter) volumes in a fermentor (Stanton and Jensen, 1993a; Mikosza et al., 1999). Alternatively, chemical reducing agents such as L-cysteine can be left out of the medium. Brachyspira species will not grow in tubes of media that have become oxidized (i.e., in which the resazurin indicator dye has become colored due to air exposure).

Fermentation and Growth Substrates

Potential fermentation substrates of Brachyspira spp. and unidentified intestinal spirochetes added to broth or solid agar media are identified as bona fide substrates when lower medium pH values (compared to medium without added substrate) result from spirochete growth (Kinyon and Harris, 1979; Jones et al., 1986; Tompkins et al., 1986; Ochiai et al., 1997). Fructose, galactose, glucose, lactose, maltose, mannose and trehalose are fermentation substrates for B. aalborgi 513AT (Ochiai et al., 1997).

The sensitivity of medium pH changes for detecting Brachyspira growth substrates is questionable. Furthermore, fermentation substrates may not always correspond to carbon and energy sources used by bacteria for growth. A more definitive method for identifying growth substrates uses a culture medium, such as HS broth, in which Brachyspira growth is limited unless a carbon/energy source is added (Table 4).

In HS broth, B. hyodysenteriae B78T, B. innocens B256T, B. intermedia PWS/AT, B. murdochii 56/150T, B. pilosicoli P43/6/78T and B. alvinipulli C1T use for growth various monosaccharides, disaccharides, the trisaccharide trehalose, and amino sugars (Table 4). Other strains of these species use similar growth substrates (Stanton and Lebo, 1988; Trott et al., 1996b). None of the tested Brachyspira strains use polysaccharides such as cellulose, hog gastric mucin, pectin, glycogen and various other tested compounds. Growth substrates for B. aalborgi have not been identified in similar experiments.

Brachyspira hyodysenteriae and B. innocens strains contain a sucrase activity that increases 3- to 10-fold when sucrose is added to cultures (Jensen and Stanton, 1994). The sucrase is the first inducible enzymatic activity to be identified for Brachyspira species.

Cholesterol and Phospholipids

Brachyspira hyodysenteriae requires cholesterol and phospholipid for growth (Lemcke and Burrows, 1980; Stanton and Cornell, 1987b; Stanton, 1997a). These requirements can be met by supplementing media with either cholesterol-phosphatidylcholine or erythrocyte membranes (Stanton and Cornell, 1987b). Cholesterol is likely required for outer membrane biosynthesis based on the following observations. Nutrient amounts of cholesterol (8– 26 nmoles/ml culture broth) are sufficient for growth (Lemcke and Burrows, 1980; Stanton and Cornell, 1987b). Brachyspira hyodysenteriae cells incorporate radiolabel from [4-14C] cholesterol into cell membrane fractions (Stanton, 1987a). Cholesterol preferentially locates within B. hyodysenteriae outer membranes (Plaza et al., 1997). The sterol requirement is unusual for a nonmycoplasma subgroup of bacterial species.

Brachyspira hyodysenteriae cellular fatty acids are distinct from those of Borrelia and Leptospira spirochetes (Livesley et al., 1993). Brachyspira hyodysenteriae and B. innocens cellular phospholipids and glycolipids were found to contain acyl (fatty acids with ester linkage) and alkenyl (unsaturated alcohol with ether linkage) side chains (Matthews et al., 1980a; Matthews et al., 1980b; Matthews and Kinyon, 1984). The culture medium for these spirochetes did not contain these same lipids, an indication that the bacteria have some capacity for fatty acid and lipid biosynthesis.

Intermediary Metabolism

Glucose and Pyruvate Metabolism

Pathways of glucose and pyruvate metabolism have been analyzed extensively only for B. hyodysenteriae (Stanton, 1989; Stanton, 1997a). The endproducts of glucose metabolism by growing cells of numerous Brachyspira species are the same as those of B. hyodysenteriae—acetate, butyrate, H2 and CO2—suggesting these species have similar catabolic routes (Stanton and Lebo, 1988; Stanton, 1989; Trott et al., 1996d; Stanton et al., 1997b; Stanton et al., 1998).

Brachyspira hyodysenteriae uses the Embden-Meyerhof-Parnas (EMP) pathway for converting glucose to pyruvate (Fig. 4). Pyruvate is catabolized by a clostridial-type clastic reaction to acetyl-CoA, H2 and CO2. Acetyl-CoA is converted to either acetate or butyrate via a branched fermentation pathway. The ATP-yielding mechanisms are substrate-level phosphorylation reactions mediated by phosphoglycerate kinase and pyruvate kinase in the EMP pathway and by acetate kinase converting acetyl phosphate to acetate (Fig. 4).

https://static-content.springer.com/image/chp%3A10.1007%2F0-387-30747-8_12/MediaObjects/978-0-387-30747-3_12_Fig4_HTML.jpg
Fig. 4.

Glucose metabolism by Brachyspira species. A) Glucose is converted to pyruvate by the Embden-Meyerhof-Parnas pathway. B) Pyruvate is metabolized by a clostridial-type clastic reaction. C) Acetyl-CoA is a branch point for pathways leading to acetate, butyrate, or ethanol. (Note: the pathway to ethanol has not been demonstrated.) NADH-H+ is oxidized in the pathways to butyrate (1) and ethanol (2) and by two alternative mechanisms: NADH oxidase (3) and NADH-ferredoxin oxidoreductase coupled to hydrogenase (4) (Stanton, 1997a).

NADH-H+ Oxidation Reactions

Brachyspira hyodysenteriae cells are versatile when it comes to NADH-H+ oxidation reactions (Fig. 4). This versatility could enhance the metabolic fitness of the spirochete in its animal host (Thauer et al., 1977; Stanton, 1997a). The NADH-H+ produced during glycolysis can be recycled or oxidized to NAD+ by 3-hydroxybutyryl-CoA dehydrogenase and butyryl-CoA dehydrogenase (butyrate pathway), by NADH-ferredoxin oxidoreductase plus hydrogenase, and by NADH oxidase (Fig. 4).

Various Brachyspira species produce more H2 than CO2, indicative of the NADH-ferredoxin oxidoreductase reaction. Ethanol is produced in cultures of B. pilosicoli, B. alvinipulli, B. murdochii, B. intermedia and B. innocens (Stanton and Lebo, 1988; Stanton, 1989; Trott et al., 1996c; Stanton et al., 1997b; Stanton et al., 1998) and is likely formed from acetyl-CoA by the enzymes acetaldehyde dehydrogenase and alcohol dehydrogenase (Fig. 4).

NADH oxidase is widely distributed among Brachyspira species (Atyeo et al., 1999; Stanton et al., 1995). The B. hyodysenteriae NADH oxidase is a water-forming, FAD-linked enzyme (Stanton and Jensen, 1993b) and its gene (nox) has been cloned (Stanton and Sellwood, 1999a). Nox-defective mutant strains of B. hyodysenteriae are sensitive to oxygen (Ecology) and lose virulence (Stanton et al., 1999b; see Virulence).

Iron Metabolism

Several observations suggest that specific iron uptake mechanisms are present and are important for Brachyspira growth in animal hosts. Brachyspira hyodysenteriae cells grow in broth containing an iron chelator, 2,2′-dipyridyl, and increase the expression of three unidentified high molecular mass proteins, >200, 134, and 109 kDa (Li et al., 1995). Catechol and hydroxamate do not enhance B. hyodysenteriae growth in iron-depleted medium, suggesting the spirochete does not use these common bacterial siderophores to bind and take up iron. Dugourd and coworkers have identified a B. hyodysenteriae genome locus, designated bit (“Brachyspira iron transport”), encoding six proteins that are likely to form an iron ATP-binding transport system (Dugourd et al., 1999). BitD has ATP-binding motifs. BitA, BitB and BitC are lipoproteins with iron binding properties. BitE and BitF resemble membrane permeases. All of the Bit proteins are smaller in molecular mass (28–42 kDa) than are previously described iron-regulated proteins (Li et al., 1995).

Genetics

Brachyspira Genes

Brachyspira genetics is at an early, promising stage of development (van der Zeijst and ter Huurne, 1997; Zuerner, 1997; Hardham and Rosey, 2000). Only a limited number of Brachyspira genes have been cloned and expressed as recombinant proteins (Table 5). Brachyspira genome libraries can be created by using E. coli-based vectors, primarily lambda phages and plasmids. An expression vector has been created that enables posttranslational processing of B. hyodysenteriae lipoprotein BlpA in E. coli and the incorporation of the protein into the E. coli outer membrane (Cullen et al., 2003). Cosmid cloning of B. hyodysenteriae DNA has generally been inefficient for unknown reasons (van der Zeijst and ter Huurne, 1997). Nearly complete 16S rRNA and 23S rRNA gene sequences are known for all Brachyspira species (Table 2) and are used for both taxonomic and clinical detection applications (See Phylogeny and Taxonomy and Identification).

Table 5.

Brachyspira genes encoding identifiable proteins.

Gene (species)

Identified/putative gene product

GenBank Accession no.

Comments

Reference(s)

gyrA (Bh)

DNA Gyrase A subunit

N/A

Partial gene sequence

Zuemer and Stanton, 1994

gyrB (Bh)

DNA Gyrase B subunit

AF288224

Not contiguous with gyrB

Stanton et al., 2001

tlyA, B, C (Bh)

Genes conferring hemolytic activity on E. coli

A31492

B. hyodysanteriae hemolysins?

Hsu et al., 2001

X73140

 

Muir et al., 1992

X73141

 

ter Huurne et al., 1992, ter Huurne et al., 1992

  

ter Huurne et al., 1994

hlyA (Bh)

Bh gene conferring hemolytic activity on E. coli

U94886

Putative amino acid sequence matches Bh hemolysin sequence

Hsu et al., 2001

hlyA (Bp)

   

Zuerner et al., 2004

nox (all 7 current species)

NADH oxidase (water-forming)

U19610, AF060800 to AF060816

Bh protection from oxidative stress

Atyeo et al., 1999

   

Stanton and Sellwood, 1999a

smpA (Bh)

Outer membrane protein

X68401

Prolipoprotein; unique to some Bh strains

Sellwood et al., 1995

   

Turner et al., 1995

bmpB (several species)

Outer membrane protein

Patent limited

Prolipoprotein; immunogenic

Lee et al., 2000

flaA

Flagellar structural proteins

X63006

Important in colonization and virulence

Gabe et al., 1995

flaB1, B2, B3 (Bh)

 

L34686

 

Koopman et al., 1993, Koopman et al., 1993

X63513

 

Li et al., 2000

AF241832

 

Rosey et al., 1996

  

Koopman et al., 1992

vspA-D (Bh)

VspH immunoreactive surface protein

AF012102

Two cluster multigene family

Gabe et al., 1998

vspE-H (Bh)

 

AY027775

 

McCarman et al., 1999

    

McCaman et al., 2003

vsh (Bh)

Capsid proteins of bacteriophage- like agent VSH-1

U90539

Putative amino acid sequences match protein sequences

T. B. Stanton et al., personal communication

mglB (Bp)

MglB homolog; immunogenic

AF200741

Putative glu-gal recognition protein (transport/chemotaxis)

Zhang et al., 2000

bitA-F (Bh)

BitA-F

U75349

Multigene system for iron transport

Dugourd et al., 1999

Unnamed (Bp)

  

Putative pyruvate oxidoreductase

Rayment et al., 1998

Unidentified (Bh)

Antigenic proteins

N/A

Uncharacterized genes and proteins; some protective

Boyden et al., 1989

tufA rpsJ (Bh)

Elongation factor Rb protein S10

U51635

N/A

T. B. Stanton, unpublished observation

blp genes G, F, E, A (Bh)

Immunoreactive lipoprotein BlpA

AY158073

Multigene family

Cullen et al., 2003, Cullen et al., 2003

bmpC (Bp)

BmpC surface protein

AY363613

Membrane vesicle lipoprotein

Trott et al., 2004

Abbreviations: VSH, virus of Brachyspira (Serpulina) hyodysenteriae; Bh, B. hyodysenteriae; Bp, B. pilosicoli; N/A, not applicable.

Genome Properties

Unfortunately, no Brachyspira genome has yet been sequenced. A physical map of the B. hyodysenteriae B78T chromosome is based on restriction enzyme fragments of the chromosome separated by pulsed field gel electrophoresis (PFGE) (Zuerner and Stanton, 1994). The B. hyodysenteriae chromosome is circular and has 3.2 Megabase pairs (Mbp), and the map (restriction fragment) locations are known for many B. hyodysenteriae genes (Zuerner and Stanton, 1994; Hsu et al., 2001; Zuerner et al., 2004). Brachyspira pilosicoli P43/6/78T has a circular chromosome approximately 2.4 Mbp (Zuerner et al., 2004) and thus carrying substantially fewer genes than the B. hyodysenteriae chromosome. Rothkamp et al. (2002) recently cloned a putative B. hyodysenteriae phosphotransferase operon that was absent from B. pilosicoli and other Brachyspira species. Differences in gene content, organization, and regulation are likely to reflect differences in host range and disease capacities among the brachyspires. The physical and genetic maps of the genomes of B. hyodysenteriae, Leptospira interrogans, Borrelia species, Treponema pallidum and T. denticola reveal broad diversity among these spirochetes in terms of chromosomal conformation (linear and circular), chromosomal numbers (1–2), size (0.95–4.9 Mbp), and number and arrangement of rRNA genes on the chromosome (Zuerner, 1997).

Plasmids and Extrachromosomal DNA

Plasmids and extrachromosomal DNAs have been reported for various Brachyspira species (Combs et al., 1989; Turner and Sellwood, 1997; Cattani et al., 1998). Unfortunately, there is little information about these nucleic acids beyond their properties in electrophoretic gels. Plasmid DNA was not detected during the construction of a genomic map for B. hyodysenteriae B78T (Zuerner and Stanton, 1994). In some reports (Combs et al., 1992; Turner and Sellwood, 1997), the characteristics of the plasmid/extrachromosomal DNAs resemble those of bacteriophage VSH-1 (virus of Serpulina hyodysenteriae; see Genetics). Some of the extrachromosomal DNAs could be DNA from VSH-1-like bacteriophage particles spontaneously produced in Brachyspira cultures.

Genetic Techniques

Mutagenesis

The first isolation of a B. hyodysenteriae strain (mutated at a specific locus, tlyA) was reported in 1992 (ter Huurne et al., 1992b). The cloned tlyA gene, conferring a hemolytic phenotype in E. coli cells, was inactivated in vitro by inserting a gene encoding kanamycin resistance at a BglII site in the tlyA gene. This construct was then introduced as a plasmid into B. hyodysenteriae cells by electroporation. Mutant cells in which the kanamycin-resistant tlyA “knockout mutation” had undergone allelic exchange with the wild-type tlyA gene were selected by plating the bacteria on media containing kanamycin (ter Huurne et al., 1992b). With some further modifications, including the incorporation of another antibiotic selection marker (chloramphenicol), this technique has been used by other investigators to create B. hyodysenteriae strains with specific mutations in genes for flagellar proteins (Rosey et al., 1995; Li et al., 2000) and NADH oxidase (Stanton et al., 1999b).

The allelic exchange technique enables investigators to “custom design” B. hyodysenteriae strains, that is, to derive mutant strains that are isogenic to their progenitor strains, except for single genetic loci. Such strains are essential in determining bacterial virulence traits (See Disease).

Brachyspira hyodysenteriae mutant strains can be produced by exposure to UV light. Using UV mutagenesis, coumermycin A1-resistant B. hyodysenteriae strains with identifiable mutations in their gyrB (DNA gyrase subunit B) genes have been generated (Stanton et al., 2001). In addition to the kanamycin resistance and chloramphenicol resistance markers used for allelic exchange mutagenesis, coumermycin resistance is a third antibiotic selection marker for genetic manipulations of Brachsypira. Some B. hyodysenteriae strains are resistant to the macrolide antibiotic tylosin, owing to a nucleotide base change in the 23S rDNA gene (Karlsson et al., 1999). Tylosin resistance can also be used as a selection marker for gene transfers in this species (T. B. Stanton, unpublished observations).

Bacteriophages and Generalized Trans-duction (VSH-1)

When cultures are treated with mitomycin C, both B. hyodysenteriae and B. innocens cells lyse and release bacteriophage-like particles whose morphology is similar to, but smaller than, λ phage virions of E. coli (Humphrey et al., 1995). These virions resemble spontaneously appearing phages first detected by electron microscopy of B. hyodysenteriae cultures (Ritchie et al., 1978). A bacteriophage was purified from mitomycin C-treated B. hyodysenteriae cultures and named “VSH-1.” The VSH-1 particles package random, 7.5-kB linear fragments of host DNA (Humphrey et al., 1997). The sequences of genes encoding several VSH-1 capsid head proteins have been deposited in GenBank (Table 5).

The significance of VSH-1 to B. hyodysenteriae genetics lies in its role as a gene vector. Purified from cultures of a chloramphenicol-resistant B. hyodysenteriae strain A203 (ΔflaA1 593:762::cat), VSH-1 virions will transfer chloramphenicol resistance to B. hyodysenteriae strain A216 (Δnox 438-760::kan) (Humphrey et al., 1997). This is the first example of natural gene transfer for a spirochete and indicates that VSH-1 behaves like a generalized transducing bacteriophage. More significantly, it is not necessary to use purified VSH-1 particles for gene exchange. Coumermycin-resistant gyrB genes are transferred by spontaneously produced VSH-1 between two B. hyodysenteriae strains when the strains are cocultured (Stanton et al., 2001).

VSH-1 may also have ecological significance. Based on MEE analysis of 231 B. hyodysenteriae strains, Trott et al. (1997c) concluded that substantial genetic recombination had shaped the overall population structure of this spirochete. Virions of VSH-1 are logical vehicles for cell-to-cell gene transfer among B. hyodysenteriae strains.

Bacteriophages have also been detected in both mitomycin C-treated and untreated cultures of weakly hemolytic intestinal brachyspires from humans and animals (Calderaro et al., 1998a; Calderaro et al., 1998b; Stanton et al., 2003). These bacteriophages should be examined for gene transduction capabilities similar to those of VSH-1.

Ecology

General Concepts

Brachyspira natural habitats are animal intestinal tracts. The species B. hyodysenteriae, B. pilosicoli, B. aalborgi and B. alvinipulli colonize the mucosal epithelial surfaces of the cecum and colon (See Habitat). Brachyspira pilosicoli and B. aalborgi have been observed attached to intestinal enterocytes (Fig. 2) whereas B. hyodysenteriae and B. alvinipulli colonize over, among, and perhaps within intestinal epithelial cells.

To be successful intestinal colonizers, all Brachyspira species must survive passage between hosts, reach suitable intestinal sites, establish dividing cell populations at those sites, and persist at least long enough to allow exiting cells to colonize other hosts. Additionally, the pathogenic Brachyspira species inflict damage on host tissues. Brachyspira ecology, knowledge of the interactions of these spirochetes with the living and nonliving components of their environment, has arisen from investigations of virulence-associated traits of the pathogenic species (Disease). Nevertheless, some of these virulence-associated traits are undoubtedly also colonization factors shared by nonpathogenic Brachyspira species. These colonization traits are discussed below.

Chemotaxis and Motility

The intestinal mucosal epithelium is covered with a layer of mucus, a barrier to bacterial colonization (Savage, 1980; Forstner et al., 1984). Brachyspira species, whether or not they attach to the underlying epithelial cells, transit and populate this mucus blanket. The ability to swim in environments of high viscosity, such as mucous gels, and the ability to be attracted to mucus components or compounds diffusing from the underlying tissues are likely to be important adaptations for Brachyspira spirochetes (Canale-Parola, 1978; Holt, 1978; Kennedy et al., 1988; Milner and Sellwood, 1994; Kennedy and Yancey, 1996). Brachyspira hyodysenteriae cells are highly motile within mucus samples taken from dysenteric pigs and do not attach to intestinal epithelial cells (Kennedy et al., 1988). Cells of the pathogen are commonly observed in mucus-filled crypts of Lieberkuhn and within mucigen droplets of mucus-secreting goblet cells (Glock et al., 1974; Kennedy et al., 1988). In chemotaxis assays, B. hyodysenteriae cells are attracted to gastric mucin, the structural glycoprotein of mucus (Kennedy et al., 1988; Milner and Sellwood, 1994). Fucose and L-serine, chemical components of mucin, are strong chemoattractants (Kennedy and Yancey, 1996).

Oxygen Metabolism, Oxidative Stress, NADH Oxidase

Those Brachyspira species that have been studied are aerotolerant anaerobes. Brachyspira hyodysenteriae cells will grow in sealed culture vessels containing an initial atmosphere of 1% O2 : 99% N2 and will consume oxygen (Stanton and Lebo, 1988). Strains of Brachyspira hyodysenteriae and of other Brachyspira species contain the enzyme NADH oxidase at high levels of specific activity (Stanton et al., 1995). The B. hyodysenteriae NADH oxidase is a soluble, FAD-dependent, monomeric protein with a molecular mass of 47–48 kDa (Stanton and Jensen, 1993b).

NADH oxidase is considered to be a mechanism by which B. hyodysenteriae cells cope with oxygen in their native microhabitats, i.e., among the oxygen-respiring mucosal tissues of the swine intestinal tract (Stanton, 1997a; Stanton et al., 1999b). Brachyspira hyodysenteriae nox-deficient mutant strains are 100- to 10,000-fold more sensitive to oxygen exposure than are cells of an isogenic, wild-type strain (Stanton et al., 1999b). The mutant stains also are attenuated in virulence (See Disease). Protection from oxidative stress is an important factor in colonization of intestinal mucosal surfaces by B. hyodysenteriae cells. NADH oxidases are involved in protecting pathogenic Streptococcus species from oxidative stress and in achieving virulence (Gibson et al., 2000; Yu et al., 2001). Brachyspira hyodysenteriae has additional enzymes for protection against oxidative stress, namely, NADH peroxidase, superoxide dismutase, and catalase (Jensen and Stanton, 1993b; Stanton et al., 1999b).

Intestinal spirochete strains representing all seven Brachyspira species (Table 1) have NADH oxidase genes and the tested strains also contain significant NADH oxidase activity (Stanton et al., 1995; Atyeo et al., 1999). Neither swine intestinal spirochete Treponema succinifaciens 6091 nor bovine rumen spirochete T. bryantii RUS-1 has detectable NADH oxidase (Stanton et al., 1995).

Attachment to Tissues and Intestinal Colonization

Brachyspira pilosicoli and B. aalborgi spirochetes attach by one end to mature intestinal cells. Colonization in this manner leads to dense parallel assemblages of spirochetes (Fig. 2). In histological examinations of sectioned intestinal tissues, these assemblages give the appearance of a false brush border. This histological phenomenon, commonly referred to as “intestinal spirochetosis,” is observed for both healthy and diseased humans and non-avian animal species (Taylor and Trott, 1997). It should be noted that the term “avian intestinal spirochetosis” (AIS) has been given to intestinal disorders of poultry associated with spirochetes regardless of whether the spirochetes are attached or not attached to intestinal tissues (Swayne and McLaren, 1997; Swayne, 2002).

For so elegant an example of prokaryotic-eukaryotic cell interactions, the mechanisms and significance of Brachyspira end-on attachments are disappointingly understudied. The polar attachment of B. pilosicoli cells to cecal enterocytes in one-day-old chicks currently provides the most practical experimental model for these investigations (Trott et al., 1995; Muniappa et al., 1998). Lattice-like structure (Sellwood and Bland, 1997) and surface, ring-like structures (Muniappa et al., 1998) observed at the tips of B. pilosicoli cells may be involved in attachment to the enterocytes.

Intestinal spirochetosis occurs in tissues from both healthy animals and animals with intestinal disorders (Takeuchi and Zeller, 1972; Neutra, 1980; Ruane et al., 1989; Barrett, 1997; Duhamel et al., 1997b). In swine this appearance is associated with B. pilosicoli-induced colitis (Taylor and Trott, 1997; Hampson and Trott, 1999b). In humans both B. pilosicoli and B. aalborgi can be detected in rectal biopsy samples where intestinal spirochetosis is identified (Hovind-Hougen, 1982; Trivett-Moore, 1998; Mikosza, 1999; Kraaz, 2000; Mikosza, 2001). Fluorescent oligonucleotide probes can be used to identify Brachyspira species among swine intestinal tissues (Jensen et al., 2000). This fluorescent in situ hybridization (FISH) technology will undoubtedly be important for the confirmed identification of spirochetes attached to cecal and colonic tissues.

Brachyspira aalborgi and B. pilosicoli may not be the only spirochete species capable of end-on attachment to enterocytes. Intestinal spirochetes attached to the rectal mucosa of humans (Neutra, 1980) and rhesus monkeys (Takeuchi and Zeller, 1972) are larger in cell diameter (0.5 µm) than cells of B. aalborgi (0.2 µm) and B. pilosicoli (0.3 µm) (Sellwood and Bland, 1997). Brachyspira-like spirochetes also colonize by end-on attachment to the cecal mucosal tissues of guinea pigs with typhlitis (Vanrobaeys et al., 1998). Other than B. aalborgi and B. pilosicoli, none of these attached spirochetes has been isolated.

Survival Outside the Host

An important aspect of the transmission of any host-associated bacterium is the ability to survive outside the host species. Brachyspira hyodysenteriae cells at concentrations sufficient to induce swine dysentery will survive up to a week in lagoon water used to clean swine buildings (Olson, 1995). Brachyspira pilosicoli cells survive in lake water for 66 days at 4°C (Oxberry et al., 1998). In laboratory studies, B. hyodysenteriae cells remain viable in diluted feces stored at 5°C for up to 60 days (Chia and Taylor, 1978). Pure cultures of B. hyodysenteriae and B. pilosicoli added at high concentrations (>109 cfu/gm) to swine feces or a 10:90 mix of feces and soil and stored at 10°C remain viable for 78–210 days (Boye et al., 2001). Environmental factors such as humidity and temperature undoubtedly affect survival. Brachyspira bacterial traits involved in survival have not been identified.

Epidemiology

Subspecies Identification

Serotype Analysis

Brachyspira hyodysenteriae strains can be subdivided into serotypes based on the immunological reactivities of lipo-oligosaccharides (LOS) in hot water-phenol extracts of whole cells (Baum and Joens, 1979; Li et al., 1991). A more elaborate system of immunological identification assigns B. hyodysenteriae strains to serogroups and subdivides the groups into serovars (Hampson et al., 1989; Hampson et al., 1997; Lau and Hampson, 1992). This system has not been widely adopted because serogroups are not entirely consistent with genetic groupings based on MEE analysis (Lee et al., 1993a; Trott et al., 1997c). Enzyme-linked immunosorbent assay (ELISA) methods based on antibodies to LOS have been used to detect animal herd exposure to B. hyodysenteriae strains (Joens et al., 1982; Wright et al., 1989).

Differentiation Based on Gene and Genome Differences

Several methods for differentiating strains of uncharacterized intestinal spirochetes and strains within the species B. hyodysenteriae or B. pilosicoli take advantage of DNA sequence differences. Sequence differences have been directly detected through restriction endonuclease analyses of genomic DNAs either with or without hybridization with specific gene probes (Combs et al., 1992; ter Huurne et al., 1992a; Koopman et al., 1993a; Harel et al., 1994; Atyeo et al., 1996; Rayment et al., 1997; Fellstrom et al., 1999).

Multilocus enzyme electrophoresis (MEE) analysis is useful for determining genetic relationships among intestinal spirochetes at the species level for taxonomy. The technique can also be used to differentiate B. hyodysenteriae or B. pilosicoli at the subspecies level for epidemiological purposes (Lymbery et al., 1990; Oxberry et al., 1998; Trott et al., 1998).

Brachyspira Population Structure

Trott and colleagues investigated the genetic diversity of 231 B. hyodysenteriae isolates by MEE (Trott et al., 1997c). The electrophoretic profiles of 12 out of 15 tested enzymes were used to identify 50 different electrophoretic types among the isolates. Based on the findings, B. hyodysenteriae appears as a genetically diverse species with an epidemic population structure. Substantial genetic recombination likely has shaped the population structure of the species. Generalized gene transduction by VSH-1 (see Genetics) could play a role in this natural genetic recombination (Trott et al., 1997c; Stanton et al., 2001). Brachyspira hyodysenteriae population structure and epidemic clones may result from disease control measures and animal management practices, including antibiotic use (Trott et al., 1997c; Fellstrom et al., 1999).

Based on both MEE and PFGE methods, population analyses of B. pilosicoli from indigenous peoples of villages in Papua, New Guinea, suggest a recombinant structure also for that spirochete (Trott et al., 1998). Thus far, assays to differentiate B. pilosicoli strains originating from different host species indicate a high diversity of genotypes for this spirochete, making it difficult to conclude that zoonotic transmission of the pathogen to humans occurs (Rayment et al., 1997; Trott et al., 1997c), although this remains a possibility.

Disease

Brachyspira hyodysenteriae, B. pilosicoli, B. intermedia and B. alvinipulli cause intestinal disease when inoculated into their normal, healthy host animals (Table 1). Brachyspira hyodysenteriae, the etiologic agent of swine dysentery, has been investigated for several decades, whereas the other two species were identified and have been studied only over the last few years. Thus, most disease-related research on Brachyspira has focused on B. hyodysenteriae.

Several recent publications extensively describe Brachyspira diseases from the viewpoint of host manifestations, clinical detection methods, therapies, and experimental models (Barrett, 1997; Galvin et al., 1997; Hampson et al., 1997; Swayne and McLaren, 1997; Taylor and Trott, 1997; Hampson and Trott, 1999b; Harris et al., 1999; Swayne, 2002; Stephens and Hampson, 2001). Published research papers from the recurrent International Conference on Colonic Spirochaetes also provide topical information on these subjects.

Swine Dysentery (B. hyodysenteriae)

Swine dysentery (bloody scours or black scours) is a severe intestinal disease that affects piglets, primarily in the postweaning stage of growth (8–14 weeks after birth). The disease has been reported worldwide in every major pig producing country. A typical sign of the disease is profuse bleeding into the large bowel lumen through lesions induced by B. hyodysenteriae cells. Afflicted animals pass loose stools containing blood and mucus and microscopically visible spirochetes. These are presumptive signs of the disease. Culturing and identifying B. hyodysenteriae cells, along with histopathological observations, provide conclusive evidence of swine dysentery. Up to 90–100% of a herd can become infected, and without effective treatment, 20–30% of infected animals may die. Economic losses result from death, poor weight gain/feed efficiency, and medication expenses. Swine management strategies, including segregation by age and prophylactic administration of antibiotics, are believed responsible for a reduction in swine dysentery in the United States in recent years.

Swine dysentery is easily produced by feeding or intragastrically inoculating normal swine with B. hyodysenteriae cultures (Kinyon et al., 1977; Kennedy et al., 1988; Stanton and Jensen, 1993a). However, the type strain B78T of B. hyodysenteriae is weakly virulent and should not be used in experimental infections (Jensen and Stanton, 1993a).

Various approaches have been used to develop whole cell or cell subunit-based vaccines for swine dysentery (summarized in Lee et al., 2000). One commercial vaccine for swine dysentery is based on pepsin-digested B. hyodysenteriae cells (Intervet, Akzo Nobel, DeSoto, KS). The immunological properties of the vaccine are now being examined (Waters et al., 2000).

Brachyspira hyodysenteriae has a limited host range. Swine are the common, but not the exclusive, hosts. Strains of the spirochete also have been isolated from juvenile rheas with a severe necrotizing typhlitis (Sagartz et al., 1992; Jensen et al., 1996; Buckles et al., 1997). Mice (Joens and Glock, 1979; Nibbelink and Wannemuehler, 1992; ter Huurne et al., 1992b; Rosey et al., 1996) and one-day-old chicks (Sueyoshi and Adachi, 1990) have been used in experimental infections. Nevertheless, nuances or inconsistencies are associated with the use of these surrogate animal models (Jensen and Stanton, 1993a; Achacha et al., 1996). For this reason, conclusions regarding B. hyodysenteriae pathogenesis based on mouse or one-day-old chick models should be confirmed through the use of swine infections.

Brachyspira hyodysenteriae Virulence Determinants

Several cell properties are putative or demonstrated virulence-associated traits of B. hyodysenteriae. They include the following: LOS in a mouse model (Nuessen et al., 1982; Nuessen et al., 1983; Greer and Wannemuehler, 1989; Nibbelink and Wannemuehler, 1991); hemolysin/hemolytic activity (Saheb et al., 1980; Saheb et al., 1981; Kent et al., 1988; Lysons et al., 1991; ter Huurne et al., 1992b; ter Huurne et al., 1994; Hutto and Wannemuehler, 1999; Hsu et al., 2001); chemotaxis/motility (Glock et al., 1974; Kennedy et al., 1988; Milner and Sellwood, 1994; Kennedy and Yancey, 1996); oxygen metabolism/NADH oxidase (Jensen and Stanton, 1993b; Stanton and Jensen, 1993b; Stanton, 1997a; Stanton et al., 1999b); and immunoreactive membrane proteins VspH and BlpA with a potential for antigenic variation (Cullen et al., 2003; Gabe et al., 1998; McCaman et al., 2003). The ability to create strains with specific gene mutations provides direct evidence of a link between virulence and NADH oxidase activity, motility/flagella (see Ecology), and hemolytic activity. Mutant strains with specific deletions of nox (Stanton et al., 1999b), flaA or flaB (Kennedy et al., 1997), or tlyA (Hyatt et al., 1994; Joens, 1997) are avirulent for swine compared to their isogenic wild-type counterparts.

Though tlyA, tlyB, and tlyC were considered to be hemolysin genes of B. hyodysenteriae (Muir et al., 1992; ter Huurne et al., 1992b; ter Huurne et al., 1994), a recent study raises questions about whether the proteins encoded by these genes are true hemolysins (Hsu et al., 2001). In that study, a protein with hemolytic activity was purified from B. hyodysenteriae cells and its gene (hlyA) was cloned in E. coli. The recombinant protein has hemolytic and biochemical activities similar to those of the native purified hemolysin. These properties are different from those of the previously described TlyA-C proteins. The latter proteins, obtained by random “shotgun” cloning of B. hyodysenteriae DNA into E. coli with selection for hemolytic colonies, are possibly B. hyodysenteriae regulatory proteins that induce synthesis of known or cryptic hemolysins in E. coli cells. Alternatively they could be “pseudo”-hemolytic proteins, the result of being over-expressed from high copy plasmids in E. coli. Interestingly, although its identity as a true hemolysin has been questioned, the tlyA gene product is essential for virulence (ter Huurne et al., 1992b; Hyatt et al., 1994). Its role in virulence should be examined further.

Spirochetal Colitis and Spirochetal Diarrhea (B. pilosicoli)

Spirochetal colitis caused by Brachyspira pilosicoli is a mild to moderate diarrheal disease of swine, birds, and possibly humans (Swayne and McLaren, 1997; Taylor and Trott, 1997; Hampson and Trott, 1999b). Watery, mucoid diarrhea and a reduction of growth rate of affected animals are common clinical signs. Spirochetal colitis of swine resembles a mild case or a very early stage of swine dysentery. The disease can be experimentally produced by inoculating pure cultures of B. pilosicoli into normal swine (Taylor et al., 1980; Trott et al., 1996c; Thomson et al., 1997).

Evidence that B. pilosicoli is a pathogen of humans is circumstantial but multifarious. Brachyspira pilosicoli strains have been isolated from humans, including some (homosexual males or those living in developing countries) with intestinal disorders and who are immunocompromised (Barrett, 1997; Trott et al., 1997a; Trott et al., 1997b). Human strains are virulent for healthy piglets (Trott et al., 1996b). A human volunteer became colonized after drinking cultures of B. pilosicoli strain Wes B (Oxberry et al., 1998). Finally, B. pilosicoli has been isolated from the blood of human patients (Trott et al., 1997b). The significance and the capacity of B. pilosicoli cells to leave the intestinal tract and circulate throughout the host’s body have not been sufficiently investigated.

Virulence factors of B. pilosicoli are unknown. By virtue of their location, outer membrane proteins undoubtedly mediate interactions between spirochete cells and their environment and are likely involved in host colonization and virulence (Trott et al., 2001; Trott et al., 2004). Cell motility, chemotaxis and spirochete end-on attachment to host tissues (see Ecology) are likely to be associated with colonization of the intestinal tract and therefore important for pathogenesis. In the absence of gross lesions, extensive colonization of intestinal tissues by B. pilosicoli cells with associated damage to microvilli could interfere with intestinal absorptive processes and lead to diarrhea (Gad et al., 1977; Taylor and Trott, 1997).

Human Intestinal Spirochetosis (B. pilosicoli, B. aalborgi, Brachyspira spp.)

There is no doubt that human colonic and rectal mucosae can be colonized by dense arrays of spirochetes (Barrett, 1997; Harland and Lee, 1967; Korner and Gebbers, 2003). The colonization is visible upon histological examination of intestinal biopsies and is known as human intestinal spirochetosis (HIS). There are unanswered questions, however, about the clinical significance of HIS and about the identities of the colonizing spirochetes. The prevalence of HIS appears greater among humans with poor living standards and among immunocompromised patients, for example HIV patients, than among healthy humans. HIS has been associated with intestinal disorders but also has been observed in healthy humans (reviewed in Barrett, 1997; Korner and Gebbers, 2003). Spirochetes in human feces and/or in association with HIS biopsies have been reported to be strains of B. pilosicoli, B. aalborgi, and unknown Brachyspira species (Brooke et al., 2003; Hovind-Hougen et al., 1982; Jensen et al., 2001; Mikosza et al., 2001). This species diversity could explain differences in clinical symptoms in humans. An alternative explanation is that HIS strains belonging to the same Brachyspira species may differ in pathogenic properties. The patient’s physiologic, immune response, and non-spirochete microbiota could also be factors in determining whether the colonizing spirochetes are commensals or pathogens. Progress in understanding HIS would undoubtedly benefit from advances in understanding the diversity and unique properties of human spirochete species.

Avian Spirochete Intestinal Diseases

Avian diarrheal diseases can have various Brachyspira species as etiological agents (Swayne and McLaren, 1997; Swayne, 2002; Stephens and Hampson, 2001, 2004). In addition to B. pilosicoli and B. hyodysenteriae (see Disease) B. alvinipulli C1T, isolated from a diarrheic chicken, is a chicken enteropathogen. The C1T cells colonize the ceca of 1-day-old chicks and 14-month-old hens and produce mild typhlitis with discolored and watery cecal contents (Swayne et al., 1995). Brachyspira alvinipulli has been isolated only from poultry and resembles an enteropathogenic spirochete earlier described by Davelaar et al. (1986).

Many studies of avian intestinal infections associated with spirochetes were made before Brachyspira species had been characterized and taxonomically established (Davelaar et al., 1986; Griffiths et al., 1987; Dwars et al., 1989). Brachyspira intermedia appears to be another common avian enteropathgen, inasmuch as spirochetes of that species have been isolated from birds with moderate intestinal colitis (Swayne and McLaren, 1997; Stephens and Hampson, 2001; Swayne, 2002). One-day-old chicks inoculated with pure cultures of an intestinal spirochete strain 1380, later identified by MEE analysis as a strain of B. intermedia (Swayne and McLaren, 1997), shed watery feces containing spirochetes and had detectable reductions in body weight compared to control birds (Dwars et al., 1992). Avian B. intermedia strain HB60 isolated from a hen with diarrhea causes reduced egg production and watery feces when inoculated into healthy hens (Hampson and McLaren, 1999a). A B. intermedia strain, freshly isolated from a swine herd with diarrhea, was not pathogenic for swine (Jensen et al., 2000).

Virulence traits of B. alvinipulli and other avian enteropathogenic Brachyspira species have yet to be determined.

Brachyspira spp. Antimicrobial Resistance

International and national guidelines for evaluating and monitoring Brachyspira antimicrobial susceptibilities under standard culture conditions are not yet available. B. hyodysenteriae strains have been divided into sensitive and resistant groups based on their susceptibilities to tylosin, erythromycin, and clindamycin (Karlsson et al., 2003). Resistance to these antibiotics is associated with a 23S rRNA gene mutation (Karlsson et al., 1999). B. pilosicoli isolates from humans or swine were found to be resistant to tetracycline, clindamycin, and amoxicillin. All strains were susceptible to an amoxicillin-clavulanic acid combination, indirect evidence that a b-lactamase is involved in resistance to amoxicillin.

Applications

Among the spirochetes, Brachyspira hyodysenteriae stands out as a good, practical choice for studying spirochete genetics and for investigating basic biological mechanisms of spirochetes, including host-pathogen interactions. The cultural, nutritional, and metabolic properties of B. hyodysenteriae have been substantially characterized and a serum-free, low-protein culture medium has been described (See Physiology). Brachyspira hyodysenteriae is an anaerobic spirochete but is aerotolerant. An experimental disease model featuring swine, the natural animal host of B. hyodysenteriae, has been available for many years (See Disease). Most importantly, recent research has provided a basis for understanding and manipulating this spirochete at the gene level (See Genetics).

Brachyspira hyodysenteriae is currently being used as a model in studies of spirochete motility. Strains with single or double mutations in specific flagellar genes are being created and used to evaluate the role of specific flagellar proteins in flagellar ultrastructure and cell motility (Li et al., 2000; C. Li and N. W. Charon, personal communication).

Footnotes
1

1The naming of uncharacterized/uncultured brachyspires is scientifically imprudent, even as a temporary precedent to proper taxonomic studies. It can become detrimental to future scientific communication. The practice is not a substitute for phenotypic and genotypic analyses of these marvelous spirochetes and should be avoided.

 

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