Morphological Heterogeneity Among Spirochetes Isolated from Cases of Swine Dysentery

Zbl. Bakt. 274, 1-15 (1990) © Gustav Fischer Verlag, Stuttgart/New York

Morphological Heterogeneity Among Spirochetes Isolated from Cases of Swine Dysentery KARl HOVIND-HOUGEN I , PETER H0GH 2, and AKSEL BIRCH-ANDERSEN 2 1

2

National Veterinary Laboratory, 1503 Copenhagen V, Denmark Statens 5eruminstitut, Depa rtment of Biophysics, 2300 Copenhagen 5, Denmark

With 13 Figures ' Received October 30, 1989 · Accepted in revised form February 27,1990

Summary Pathogenic and non-pathogenic spirochetes isolated from the intestines of pigs were examined by electro n microscop y using the negative staining and ultrathin sectioning techn iques. Morphological differences were observed among cells of different stra ins. The cells differed in length as well as in width and in the number of flagella inserted at each end . In addition, the cells from different strains also varied in their resistance to the action of the detergents, Teepol and sodium deox ycholate . Three of the strains studied contained weakly haemol ytic spirochetes, two of which differed markedl y in their morphology from the cells of the other strains. These spirochetes had fewer flagella inserted at each end tha n tho se from other isolates and showed a distinct lattice-like substructure covering the ends of the cells. The spirochetes examined were found to be morphologically more similar to those of the genus Borrelia than to those of the genus Treponema but were clearly different from the cells of both of these genera . The taxonomic implicati ons of the observations are discussed in brief.

Zusammenfassung Aus dem Darm von 5chweinen isolierte path ogene und nicht-pathogene Spirochaten wurden mit Hilfe der Verfahren der Negativkontrastierung und des Ultradiinnschnitts elektronenmikroskopisch untersucht. Es wurden morphologische Unterschiede zwischen den Zellen vcrschicdener Stiimme beobachtet. Die Zellen unterschieden sich nach Lange und Breite sowie der Zahl der an den Enden ansetzenden Gei~eln. Dariiber hinaus zeigten die Zellen von verschiedenen Stammen auch Unterschiede in ihrer Resistenz gegen die Wirkung der Detergentien Teepol und Natriumdeoxycholat. Drei der untersuchten Starnme enthielten schwach hamolytische Spirochaten, von denen sich zwei deutlich in ihrer Morphologie von den Zellen anderer Starnme unterschieden. Bei diesen Spirocharen sa~en weniger 1 Zbl. Bakt. 27411

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K. Hovind-Hougen, P. Hegh, and A. Birch-Andersen

GeitSein an den Enden als bei den Zellen aus anderen Isolaten, und sie zeigten eine deutliche Gitter-Substruktur an den Zellenden. Es wurde festgestellt, datS die untersuchten Spirochaten morphologisch mehr denen des Genus Borrelia als denen des Genus Treponema ahnelten; sie unterschieden sich aber deutlich von den Zellen beider genannten Genera. Die sich daraus ergebenden taxonomischen Foigerungen werden kurz behandelt. Introduction Spirochetes have, for many years, been known to be involved in swine dysentery. Their role in th is disease was, however, uncertain until Taylor and Alexander (15), and Harris and co-workers (4 ) in 1971-72 published evidence demonstrating that these microorganisms are the causative agent of swine dysentery. Non-pathogenic spirochetes are often isolated from presumably healthy pigs and these organisms are morphologically identical to the pathogenic ones when examined by light microscopy. In addition to differences in enteropathogeneity, these spirochetes can also be distinguished by differences in their haemolytic activity, whe re pathogenic spirochetes show a strong haemolysis and non -pathogenic ones are only weakly haemolytic. However, in 1980 Taylor et al. isolated a weakly haemolytic spirochete which produced diarrhoea in pigs and which differed morphologically from the organisms of previous isolates by being thinner and by possessing onl y five flagella inserted at each end (16 ), in cont rast to earlier isolated ones which were described to possess 8-9 (1 1). This last mentioned isolate also differed from those previously described when cultured and consequently it was considered to belong to another species than T.

hyodysenteriae ( 16). In 1979 Kinyon and Harris published their results of measurements on cells of pathogenic and non -pathogenic T. hyodysenteriae and demonstrated a larger variation

among cells of the same strain than previously observed in strains of other spirochetes (11 ). In order to see if similar results were obtained with Danish isolates, we performed a preliminary study of the morphology of cells of a few Dan ish isolates of spirochetes obtained from pigs with swine dysentery, and variation in cell dimensions as well as in the number of flagella were observed. Consequently, it was decided to perform a more extensive study of the ultrastructure of the cells of some of the strains of T. hyodysenteriae which are often used as reference strains in research projects on swine dysentery, and which originally had been isolated from diseased animals in different countries. The results of these studies are described in the present paper and their implications on the taxonom y of these spirochetes is discussed.

Materials and Methods

Strains The strongly haemolytic strains used were B78, type strain of T. hyodysenteriae, B204, and B169 all obtained from]. Kinyon, College of Veterinary Medicine, Iowa State University, Ames, Iowa, USA; PI8-A obtained from Dr. R. Lemcke, Compton, England, and Dys 754 isolated in Denmark. The pathogenicity of these strains was confirmed (10, 12 and Hegh, unpublished data ). Two non-pathogenic strains were included, B256, the type strain of T. innocence (11) and Dys 40-3 (Hogh, unpublished data). Furthermore, a weakly haemolytic, pathogenic strain P43-6 (16) also obtained from Dr. R. Lemcke was included.

Morphology of Swine Intestinal Spirochetes

3

Cultivation

All strains were routinely maintained on trypticase soy agar plates supplemented with 5% bovine blood, 400 ug/ml spectinomycin and 5 !-tg/ml polymyxin, except for B256 which was maintained on plates without spectinomycin and polymyxin. The cultures were incubated at 41.5°C in an atmosphere of 50% CO 2 and 50% H 2 for three days. Strains B78, B256 and P43-6 were also cultivated in trypticase soy broth (TSB) supplemented with 10% calf serum and glucose at 42 °C in an atmosphere of 80% CO 2 and 20% H 2• Preparations for electron microscopy Negative staining . Cells were washed off the plates with about two ml of a 0.03% sucrose solution to which were added 0.01 M MgCl 2 and 0.01 M CaCl 2 (SMC). Specimen grids were prepared by the multiple drop technique (8). Cells of some strains were further treated on the grids with 0.2% Teepol or 2% sodium deoxycholate, both in redistilled water. All preparations were negatively stained with 1% ammonium molybdate, pH 7. Ultrathin sections . Liquid cultures of the strains B78, B256 and P43-6 were centrifuged at 8000 x g for 20 min. One pellet of each strain was suspended in a 1 : 1 mixture of TSB and SMC. One ml of 3% glutaraldehyde in SMC was added to two ml of cell suspension and the cells were fixed at room temperature for 45 min. The suspension was centrifuged at 8000 x g for 20 min. The pellet was taken up in a few drops of 1.5% warm (45°q, melted Noble Agar Difco in SMC. Small blocks with visible cell clusters were cut and fixed overnight in 1% osmiumtetroxide in SMC with 10% TSB added. Another pellet of each strain was suspended in 2 ml 3% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2 (CB) + 2 ml CB + 2 ml 0.15% ruthenium red in redistilled water (RR), and fixed at room temperature for 2 h. The suspensions were centrifuged at 8000 X g for 20 min and the resulting pellets resuspended in a mixture of 1 mil % OS04 in CB + 1 ml CB + 2 ml RR, and fixed overnight at room temperature. The suspensions were centrifuged and the pellets were taken up in agar as above. Both preparations were treated with 2% uranyl acetate in redistilled water for one hour, dehydrated in alcohol and propyleneoxide prior to embedding in Vestopal-W. Sections were cut on a LKB ultrotome III, and stained with magnesium uranyl acetate (3) and lead citrate (14).

Electron microscopy

Electron microscopy was carried out on a Philips EM 200 electron microscope at primary magnifications of 1400, 3000, 9000 and 16000 x. Organisms lying approximately at or in the centre of a grid square of a 400 mesh grid, so that their entire length could be measured, were photographed at either 1400 or 3000 x. For this purpose, only one exposure was made in each square. Enlargements were made of all cells that appeared undamaged and regularly waved. Subcellular structural details were photographed at either 9000 or 16000 x . Negatives were obtained on Kodak Fine Grain Release Positive Film, Type 5302, and were photographically enlarged 10 times. Measurements and calculations

The length of the cells was measured along the axis of the helices on prints with a total magnification of 14000 or 30000 x (Figs. 1 a, b) The number of waves per cell was counted and the wavelength calculated . The width was measured approximatealy in the central part of a cell. The amplitude was measured as half the distance between the top and the bottom of individual waves. In the present investigation, about 600 micrographs were studied .

4

K. Hovind-Hougen, P. Hegh, and A. Birch-Andersen Statistics

Arithmetic mean values were calculated for length, width and wavelength of the cells of each strain. The standard deviation did not vary much from strain to strain and standard errors were, therefore, based on pooled estimates of the variance within strains. Furthermore, Kruskal-Wallis tests were used to test whether the strains could all have the same distribution by length, width and wavelength. Results Cells of all strains were regularly waved (Figs. 1 a, b), except those of strain B256 which often formed a loop or were curled up in a spiral. All cells narrowed at their ends, but the actual end of the cell was blunt (Figs. la, 2). Cells of the strains Dys 40 -3 and P43-6 had a characteristic long, narrow extension of the cell ends, which on most cells was covered by a surface layer with a regular substructure (Fig. 3). The helical cells were generally between 5 and 12 urn long. Cells of stra in Dys 754 were the shortest of all the strains tested. The shortest cells of the weakly haemolytic strain B256 were shorter than the shortest cell of Dys 754, but the longest cells of B256 were much longer than the longest ones of Dys 754 (Table 1). The cell diameter varied between 0.30 and 0.35 urn for cells of the strongly haemolytic strains, and between 0.25 to 0.30 urn for the weakly haemolytic ones (Table 1). The

1a 1b

Figs. la and b. Regularly waved cells of strain P18a (a) and Dys 40-3 (b). Note that the ends of the cell in Fig. 1b is more tapered than those on the cell in Fig. 1a. Negatively stained with 1% ammonium molybdate. x 15000. Bar, 1 urn.

Mor phology of Swine Intestinal Spirochetes

5

Ta ble 1. Morphological and haemolytic characters of cells of strains of spirochetes isolated from swine intestines Strain

B78 B204 B169 Dys 754 P18A B256 Dys 40-3 P43-6

Length * range

Width * range

Wavelength "

No of flagella

Width of flagellar shaft" * sheathed naked

Haemolysis

6.6-10.8 6.6-25 .3 6.4-17.2 5.0-6.6 5.7-15.3 6.0-11.7 4.8-9.9 5.1-10.1

0.27-0.35 0.30-0.35 0.29-0.37 0.32-0.35 0.28-0.40 0.20-0.35 0.26-0.33 0.20-0.27

2.0-5.5 2.0-3.6 3.0-3.9 2.1-3 .6 2.3-5 .0 2.9-4.8 2.6-4.0 2.2-4.6

12.- 14 (7) 8 (8) 13 (9) 10 (9) 10 (9) 13 (13) 5 (14) 5 (11)

20 (20) 22 (6)

14 (9) 16 (3)

17 16 19 21 21

11 11 14 15 13

Strong Strong Strong Strong Strong Weak Weak Weak

range

(13) (4) (11) (5) (7)

(10) (4) (5) (5) (7)

* All dimensions are in urn * * All dimensions are in nm For length, width and wavelength the number of observations is the same as stated in Table 2. Numbers of observations for the flagella are given in brackets .

Ta ble 2. Arithmetic mean values Strain B78 B204 B169 Dys 754 P18A B256 Dys 40-3 P43-6 B78+B256,x,n Standard deviation P

x

Length n

x

Widt h n

Amplitude n

x

Wavelength n x

7.6 11.1 12.9 5.9 8.1 8.8 6.7 7.3

9 9 11 7 11 11 8 9

0.29 0.33 0.35 0.34 0.36 0.25 0.29 0.23

9 9 11 7 11 10 8 9

1.16 0.87 1.12 1.01 1.21 1.11 0.88 0.73

3 8 5 6 8 7 7 6

2.91 3.24 3.32 1.90 3.36 3.56 3.07 3.18

7 7 10 7 11 11 8 7

8.2

20

0.27

19

1.12

10

3.31

18

2.89

0.413

0.251

7.63

0.00007

1.7 x 10-7

0.028

0.36

x is the arithmetic mean in urn, There is no indication of a skew distribution of observations

and consequently the calculations are based on the arithme tic mean and not the logarithmic. P is the p value for a Kruskal-Wallis test (non-parametric test) to establish whether the strains could all have the same distribution according to the parameters measured. Because there is no difference between the strains B78 and B256, a common mean has been calculated for these strains .

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K. Hovind-Hougen, P. Hegh, and A. Birch-Andersen

3

Fig. 2. End of a Dys 754 cell. The insertions of the flagella, F, are arranged in a V-shaped formation (arrows). SL denotes surface layer. Some loose flagella are also present, F. Negatively stained with 1% ammonium molybdate. X 90000. Bar, 100 nm. Fig. 3. End of Dys 40-3 cell showing the characteristic extension on which the surface layer (SL) presents a network-like pattern. F denotes flagella. Negatively stained with 1% ammonium molybdat e. x 90000. Bar, 100 nm.

Morp hology of Swine Intestinal Spirochetes

7

wavelengths of th e cells were 3.0-3.5 11m and were rather simila r for all strains (Table 2). The amplitude was about I 11m and also rather similar for cells of all th e strains examined (Table 2). A comparison of the strains on the basis of lengt h and width of the cells is shown in Fig. 4. A reasonable overlap for both length and width is required for the strains to be iden tica l. From the da ta obtained it can be seen that strains P18A and Dys 754 could be identical; and so cou ld the strains B256, B78 and pro bab ly also Dys 40-3 . Furthermore, the cells of strains B169 and B204 could also be identical on the basis of these obse rvations. At a higher magni fication , the majority of the cells were seen to be covered by a surface layer (Figs. 2, 3 and 6) which on some micrographs showed a regu lar substructure (not shown). A characteristic network-like pa ttern was seen on the extension at the tips of th e cells of the strains Dys 40-3 and P43-6 (Fig. 3). This pa ttern was not observed along the main part of the cells. All cells had a bundle of flagella inserted at each end, and the flagella were wound around the cytoplasmic bod y of the cells. The flagella overlapped in the central regio n of the cells. Cells of the str ains B78, BI69 and B256 had 13 flagella inser ted at each end. The insertion points of the flagella seemed to be arranged in a V-shaped configuration within the cells of all strains (Fig. 2) except for those of Dys 40 -3 and P43-6 which showed five flagella at each end inserted in a row along the longitudinal axis of the cell (Fig. 3). Cells of P18A and Dvs 754 had ten flagella inserted at each end with the insertion points distributed in a Vvshaped pattern as for the cells with 13 flagella (Fig. 2). Cells of strain B204 had eight flagella inserted at each end .

4

Comparison of length and width of Spirochetes isolated from swine intestines Width in JM1l

0,4

i

i

•

J

754

1 i

0,3

1•!

B 169

l

B204 •

40 -3

i

f 1

P l8A

8 78 { B 2 56 -

P4 3-6 I

0,2

+ length inJM1l

3

4

5

6

7

8

9

10

11

12

13

14

15

Fig. 4. The lines show the 95% confidence interval for length (horizontal) and width (vertical). The correlation between the measurements is not so prono unced that it should be taken into consideration . A moderate overlap for both length and width is necessary in order to prove that two strains are identical.

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K. Hovind-Hougen , P. Hegh, and A. Birch-Andersen

Dividing cells with two cells present within a mutual outer envelope were occasionally seen. The plasma membranes of the "new" cells at the site of division were arranged at right angles to the outer envelope of the mother cell (Figs. 5 and 13). Flagellar insertion points were seen on both sides of the site of the binary fission which leads to daughter cell formation . Some flagella from the ends of the mother cell could be seen to pass across the division site (Fig. 5). Many cells from the liquid cultures of B78 and B256 appeared to be fragile and were more or less damaged during preparation for electron microscopy so that free flagella and cellular debris were found together with apparently undamaged cells. Occasionally, apparently undamaged surface layers enclosing only flagella were observed in these cultures (Fig. 6). Cells of some strains were treated on the grid with the detergents Teepol or sodium deoxycholate. For most of the strains, the surface layer and the outer envelope of the cells were destroyed after treatment with Teepol for up to two minutes, and only flagella and cytoplasmic bodies were left (Fig. 7). However, in the cells of strain B256

Fig. 5. Part of a dividing cell of B169. The two daughter cells are within a mutual outer membrane (arrow). The cell ends are truncated at this stage of division. Arrowheads denote insertion points for flagella. Some flagella (F) from the parent cell are seen to pass the division site. Negatively stained with 1% ammonium molybdate. x 90000. Bar, 100 nm. Fig. 6. Only the surface layer (SL) is enclosing the flagella (F) of this cell in a preparation of B 256 cells grown in liquid medium. Negatively stained with 1% ammonium molybdate. x 45000. Bar, 0.5 urn.

Morphology of Swine Intestinal Spirochetes

9

7

I

Fig. 7. Flagella and the cytoplasmic body are left after treatment of cells of Dys 754 with 0.2% Teepol for one min. Note the unsheathed part of some flagella (arrows). Negatively stained with 1% ammonium molybdate. x 90000. Bar, 100 nm. Fig. 8. Part of a cell of B256 after treatment with 0.2% Teepol for two min. Only flagella and a damaged cytoplasmic body are left. The basal bodies (B) and the hooks (H ) are seen on some of the flagella. Negativel v stained with 1% ammonium molybdate. x 90000. Bar, 100 nm.

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K. Hovind-Hougen, P. Hogh, and A. Birch-Andersen

9a

o

9b

10

Fig. 9a and b. Details of flagella loosened from cells of Dys 754 which had been treated with Teepol for one minute . Note the individual elements of the basal complex of the flagella : disc (D), collar (C) and hook (H) . Negatively stained with 1% ammonium molybdate. x 160000. Bar, 100 nm. Fig. 10. Cell of B 256 treated with 2% sodium deoxycholate for two min. Flagella (F) and membranous debris (M ) are left. and discs (D) and hooks (H ) are present on of some of the flagella. Negatively stained with 1% ammonium molybdate. x 90000. Bar, 100 nm.

12b

12c R

Figs. 11, 12a, band c. These figures show sections of cells after fixation and embedding as described in the text . The cells are surrounded by a triple-la yered outer membrane (OM ) and a triple layered cytoplasmic membrane (CM), both of which are asymmetric with the outer dense layer being thicker and more electron-dense than the inner one. Flagella (F) are situated between the outer membrane and the cytoplasmic membrane (CM). Note that two rows of flagella (arrows) are present in some cross-sectioned cells. The cell interior is densly packed with ribosomes (R). Stacks of membranes (arrowheads) as seen in Fig. 12a were observed in a few cells of strain B78. Figs. 11 and 12a present cells of strain B78, Figs. 12b and 12c cells of strains B56 and P43-6 , respectively. Cells shown in Figs. 12a, band c were fixed with ruthenium red present in the fixative. x 90000. Bar, 100 nm.

12

K. Hovind-Hougen, P. Hogh, and A. Birch-Andersen

also the cytoplasmic bodies were damaged by this treatment (Fig. 8). After the Teepol treatment, many flagella were detached from the cytoplasmic bodies of the cells and on these, the flagella basal complex was seen to be similar to that present on flagella detached from treponemes and borrelia as well as from gram-positive bacteria, i.e. a pair of discs connected by a narrow neck to the hook of the flagellum (Figs. 9a, b). The flagellar shaft was surrounded by a sheath, the length of which was shorter than the length of the shaft, thus leaving the distal part of the flagellum unsheathed. The width of the shaft with sheath included was about 20 nm and that of the core was 14 nm for flagella isolated from cells of all strains studied (Fig. 8) except for those of Dys 754 and P18A where the dimensions were 17 nm and 11 nm, respectively (Fig. 7). The distal parts of flagella which were detached from cells after accidental damage during preparation for electron microscopy were also found to be unsheathed and so were those detached from cells treated for 2 min with 2% sodium deoxycholate. Tubules of the kind found to be present in the interior adjacent to the cytoplasmic membrane of cells of Treponema species (7) were never observed, neither in detergent-treated nor in accidentally damaged cells of the present material (Figs. 6 and 10). In our ultrathin sections, the cells appeared to be better preserved when ruthenium red had been added to the fixative. However, spheroids were seen in all preparations. Cells of all strains were surrounded by an outer triple-layered membrane of which the outer dark leaflet was thicker and more electron-dense than the inner one (Figs. 11, 12a, b, c). A surface layer exterior to the outer leaflet was more pronounced on cells of B78 than on tho se of B256 and P43-6 (Figs'. 12a, b, c). In the cells of all strains studied, the cytoplasmic membrane appeared slightly asymmetrical with the outer leaflet being darker and more electron-dense than the inner one (Figs. 12a, b, c). The asymmetry of the cytoplasmic membranes was less pronounced in cells of the strains B256 and P43-6 (Fig. 12a and Figs. 12b, c). The flagella were situated in the interspace between the cytoplasmic membrane and the outer envelope and were observed in cross-sectioned organisms in either one or two rows depending on the part of the organism that was included in the section (Fig. 11). The cytoplasm was so densely packed with ribosomes, that regions with DNA filaments could not be resolved. Stacks of membranes were observed in the cytoplasm of only a few cells of strain B78 (Fig. 12a). A few fortunate sections showed the division site of dividing organisms. The asymmetry of the cytoplasmic membrane was very pronounced on the new ends formed in the middle of the cells (Fig. B ).

13 Fig. 13. Part of a section of a dividing cell of strain P 43-6. Note the truncated ends of the daughter cells and the pronounced asymmetry of the cytoplasmic membrane (e M ). x 90000 . Bar, 100 nm.

Morphology of Swine Intestinal Spirochetes

13

Discussion

Kinyon and Harris studied the dimensions of several spirochetes isolated from pig intestines and concluded that they were unable to find any statistically significant differences between the sizes of the cells of pathogenic and non-pathogenic strains of T. hyodysenteriae (11). The wavelengths and amplitudes determined for the cells of our material were found to be rather similar for cells of all strains examined and consequently, these parameters were of no use for differentiation between strains. The variation in cell length as well as in cell width could, however, together with other morphological characters, be used to distinguish between different strains. Based on the length and width of the organisms, cells of the strains B78 and B256 appeared to be identical, and those of Dys 40-3 to be very similar to them. Cells of strains B78 and B256 were also identical with respect to the shape of their cell ends and the number of flagella inserted at each end. However, cells of strain B256 tended to form loops or spirals which B78 cells did not. Furthermore, the cells of these two strains showed a different tolerance to treatment with detergent and the surface layer was more extensive on sectioned cells of strain B78 than on those of B256. It thus appears that the cells of these two type species were morphologically identical but differed in their reaction to detergent and ruthenium treatment . Cells of strain Dys 40-3 differed in three respects from those of B78 and B256; 1) the ends of the cells were more pointed, 2) the ends possessed a regular network-like substructure and 3) only five flagella inserted in a row were present at each end of the cells. These 3 characteristic differences shown by cells of the Dys 40-3 strain were shared also by those of strain P 43-6 . Also the lengths of the cells of these two strains were similar, but their widths were clearly different with the P 43-6 cells being thinner than the others (Tables I and 2). Cells of the strains B204 and B169 had approximately the same length and width but differed by the number of flagella inserted at their ends. Cells of the strains Dys 754 and P18A were morphologically very similar. They possessed the same number of flagella and their flagella were thinner than those of the other cells examined. Previous studies on spirochetes have shown that cells with different numbers of flagella also show differences in other parameters studied and thus belong to different species (2). Cells with the same number of flagella inserted at each end may be identical, but may also very well be so different that they even belong to different species (2). When the results of the present morphological study were compared to those of Baum and Joens (1) on the precipitin reactions of hot-phenol water extracts of pathogenic T. hyodysenteriae with immune sera from rabbits, we found full agreement with the results concerning strains B78, B204, B169 and PI8A. We found that the cells of these strains were morphologically different and the precipitin tests showed that they all belonged to different serogroups (1). None of the cells examined in the present study contained the cytoplasmic tubules which are regarded as a morphological criterium for the classification of treponemes (6, 7). Furthermore, the cells divided with a formation of septa similar to cells of the genus Borrelia, and not with a formation of constrictions like treponemes. Prior to the isolation of the spirochetes causing dysentery (5, 15), the spirochetes isolated from healthy or diseased pigs were called Borrelia-like because they were thicker than treponemes, rathe r loosely coiled and easily stained with aniline dyes (9). Harris and co-workers, however, classified the spirochetes as belonging to the genus

14

K. Hovind-Hougen, P. Hogh, and A. Birch-Andersen

Treponema on the basis of their morphology, their location in the animal host and anaerobic requirements (5, 11). However, the cells of the strains examined in the present study are morphologically different from all previously examined strains of the genus Treponema (7). On the other hand, ther e are many similarities between the morphological characters of T. hyodysenteria, T. innocens and Borrelia, but there are also obvious differences. The gross morphology of T. hyodysenteriae and T. innocens is very different from that of Borrelia. One can never mistake a T. hyodysenteriae for a Borrelia by examination under the light microscope. Cells of borreliae appear longer and thinner than those involved in swine dysentery and their wavelengths seem shorter. Furthermore, Borrelia are known to be transmitted by vectors , and up to the present time, no vector for the transmission of T. hyodysenteriae has been established. Paster et al. (13) studied the phylogeny of spirochetes by the RNA oligonucleotide cataloging method and found B. hermsii, the only borrelia included in the study, to represent a deep branching in the line of descent that gave rise to the main spirochete cluster. In contrast, T. hyodysenteriae was as distant from the main spirochete-treponeme group as leptospires and it was concluded that T. hyodysenteriae may represent an entirely new undesignated spirochaetal genus. The results of the present work on the morphology of cells of T. hyodysenteriae support this conclusion. From our results on cells of strains P43/6 and Dys 40-3 , these two strains should be considered as belonging to another species than T. hyodysenteriae and T. innocens (16) within such a new genu s. Further work is in progress on the heterogeneity of spirochetes isolated from swine intestines and for comparison of these to Borrelia isolated from different sources. Acknowledgement. Our thanks are due to cando stat. Severin Olesen Larsen, Statens Seruminstitut, for performing the statistical analysis. We also wish to thank Birgit Ottesen for technical assistance, Elisabeth Brakti for sectioning and electron microscopy, and Anne GreteOvergaard and Evert Muller for the photographic work. Tove Fabricius, KettyMoller and Hanne Lane are given our thanks for skilfull secretarial assistance. - The work was supported by Grant No. 513-20012 from the Danish Agricultural and Veterinary Research Council.

References 1. Baum, D. H. and L. A. Joens: Serotypes of beta-hemolytic Treponema hyodysenteriae. Infect. Immun. 25 (1979) 792-796 2. Fiehn, N. E.: Small-sized oral spirochetes and periodontal disease. APMIS 97, Suppl. 7 , (1989) 4-3 1 3. Frasca, j. M. and V. R. Parks: A routine technique for double-staining ultrathin sections using uranyl and lead salts. ]. Cell BioI. 25 (1965) 157-161 4. Harris, D. L., j. M. Kinyon, and R. D. Glock: Isolation and propagation of spirochetes from the colon of swine dysentery affected pigs. Can.]. CompoMed. 36 (1972) 74-76 5. Harris, D. L., R. D. Glock, C. R. Christensen, and J. M. Kinyon: Swine dysentery. 1. Inoculation of pigs with Treponema hyodysenteriae (new species) and reproduction of the disease. Vet. Med. Small Anim. Clin, 67 (1972) 61-64 6. Hovind-Hougen, K.: Morphology . In: R. F. Schell and D. M. Musher (eds.), Pathogenesis and Immunology of Treponemallnfection, pp. 3-28. Marcel Dekker, New York (1983)

Morphology of Swine Intestinal Spirochetes

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7. Hovind-Hougen, K.: Determination by means of electron microscopy of morphological criteria of value for classification of some spirochetes, in particular Treponemes. Acta path. microbiol. scand. B, Suppl. 255 (1976) 8. Hovind-Hougen, K. and A. Birch-Andersen: Electron microscopy of endofiagella and microtubules in Treponema Reiter. Acta path. microbiol. scand. B 79 (1971) 37-50 9. ]anitschke, P. und P. Kielstein: Moglichkeiten zur Diagnostik und Bekampfung des Schweinedysenterie. Mh . Vet.-Med . 31 (1976 ) 721-726 10 . Kinyon,]. M., D. L. Harris, and R. G. Glock: Enteropathogenicity of various isolates of Treponema hyodysenteriae. Infect. Immun . 15 (1977) 638-646 11. Kinyon, ] . M. and D. L. Harris: Treponema innocens, a new species of Treponema hyodysenteriae intestinal bacteria, and emended description of the rye strain of Harris et al. Int. J. System. Bact. 29 (1979) 102-109 12. Lemcke, R. M. and M. R. Burrows: A comparative study of spirochetes from the porcine alimentary tract. J. Hyg. (C arn b.) 86 (1981) 173-182 13. Paster, B. ] ., E. Stackebrandt, R. B. Hespell, G. M. Hahn, and C. R. Woese: The phylogeny of the spirochetes. System. Appl, Microbiol. 5 (1984) 337-351 14. Reynolds, E. 5.: The use of lead citrate at a high pH as an electron opaque stain in electron microscopy. J. Cell BioI. 17 (1963) 208-212 15. Taylor, D. j. and T. j. L. Alexander: The production of dysentery in swine by feeding cultures containing a spirochete. Br. Vet. J. 127 (1971) 58-61 16. Taylor, D. ] .,]. R. Simmons. and H. M. Laird: Production of diarrhoea and dysentery in pigs by feeding pure cultures of a spirochaete differing from Treponema hyodysenteriae. Vet. Rec, 106 (1980 ) 32(,-.B2 Dr . Kari Hovind-Hougen, National Veterinary Laboratory, P. O. Box 373, DK-1503 Copenhagen V, Denmark