In vitro attachment of Treponema hyodysenteriae to mammalian epithelial cells

Dcl~ot ' t t~ let~t c~/"Metlictrl M icroh io log~ , Cr.eigl~totr Urrrcer'.siry Srlrool ofiCletlicitre. 0111trllt1, NE, U . S . A . 68/78

GORDON D. SCHRANK Dc,~)ortr?rc,t~r c!l'Microbiolog~, Ut l i~er .~ i ty of Tet~tre.s.ree Cetlter fbr tlle Herrltlr Scir t~ces, M t t ~ r p h i . ~ , T N , U . S . A . 38/63

AND

FRANK M. FERRARO Deprrrttt~rt~t c~j'Mctlirrrl Microbiology, C~.eiglrtor~ Ut~iuersiry Scllool of Metiic,itre, Ottltrl~rr, NE, U . S . A . 68/78

Accepted November 22. 1978

KNOOP, F. C . , G. D. SCHRANK, and F. M. FERRARO. 1979. In vitro atrachment of Trc,potrrtrlrr h.votlysc,t~terirre to mammalian epithelial cells. Can. J . Microbiol. 25: 399-405.

The interaction between Trepot~et~itr l~yorl~~.srt~terirre and isolated swine intestinal epithelial cells or mouse adrenal cells in culture was examined. Studies were performed in which tre- ponemes were incubated with each type of animal cell in an atmosphere of 5Yo CO, in air-. Coincubation was te~minated at v;rl.ious time intervals, and the percentage of treponemal attach- ment evaluated by light microscopy. The extent of attachment wasdependent on both incubation time and tempelxture. The mechanism of attachment to the animal cell surface wasexamined by scanning electron microscopy. Intel-action of the parasite with the host cell did not appear to alter cellular morphology or result in changes of the cell surface at the site of attachment. Preference for a cellular site of attachment was not found.

KNOOP. F. C., G. D. SCHRANK et F. M. FERRARO. 1979. In vitro attachment of Trcpotretnrr l~yor!,..spt~terirre to mammalian epithelial cells. Can. J . Microbiol. 25: 399-405.

On aetudie I'interaction entre Trrpotlet71tr Ir.vorIyset~tr~irre et des cellules isolees de I'epithelium intestinal du porc ou des cellules en culture de surrenales de souris. Ces etudes ont ete effectuees en incubnnt les trepontrnes avec chacun des types de cellules animales dans une atmosphere contenant 5% de C 0 2 . Cette incubation mixte bacteries-cellules a ete arretee i differents inter- valles et le pourcentage d'attachement cellulaire du tl-eponeme a ete mesure en rnicroscopie ordinuire. Le degre d'attachement depend du temps d'incubation et de la temperature. Le mecanisme de la fixation bactkrienne B la surface de lacellule zrnimale a I te etudie en rnicroscopie :I balayage. Cette interaction du parasite avec lacellule-h6te ne semble pas alterer la rnorphologie cellulxire ni modifier la surfirce cellulaire au site d'attachement. Cette fixation de la bacterie ne semble pas s'effectuer B un site cellulaire en pirrticulier.

[T~xduit par lejournal]

Introduction Extensive evidence has accumulated on the

etiology of swine dysentery (SD), a mucohemor- rhagic diarrheal disease of swine. The disease is characterized by inflammation, excess mucus production, and necrosis of the large intestine. These manifestations often result in frank hemor- rhage, dehydration, and eventually death (Dunne and Leman 1975). Although SD was first described by Whiting et 01. (1921), the etiology of the disease was not suggested until 197 1 (Taylor and Alexander 1971 ; Taylor and Blakemore 1971). In 1972, a tr-e- poneme was isolated in pure culture and found to induce the classical signs and symptoms of SD

when orally inoculated into specific pat hogen-free swine. The organism was further characterized and named T~.eponemer hyocbsentericre (Harris et rrl. 1972).

Much research remains to fully understand the complex disease processes operative in swine dysentery. The lack of an alternate in vivo animal system, or in vitro methods to investigate attach- ment and colonization of the causative agent, has hindered attempts to investigate virulence deter- minants of the organism.

In this report the attachment of T. hyodysen- tericre to isolated swine intestinal epithelial cells or mouse adrenal cells in culture was examined. In- vestigations were conducted to determine optimal conditions for attachment of the organisms to the

'Address reprint requests to Dr. Knoopat the Department of host cell su~face and to study the interaction of T. ~ ~ ~ l i ~ ~ l ~ i ~ ~ ~ b i ~ l ~ ~ ~ , creighton university school of ~ ~ d i . hyodysetlterirre with cultured cells by scanning cine, 2500California Street, Omaha, NE. U.S.A. 68178. electron microscopy.

0008-4 166/79/030399-07$01 .OO/O 01979 National Research Council of CanadalConseil national de recherches du Canada

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400 CAN. J . MICROBIOL. VOL. 25. 1979

Materials and Methods A continuous cell line of mouse Y I adrenal cells was obtained

from S. T. Ilonta (University of Iowa and Vetefi~ns Administra- tion Hospitals. Iowa City. [A. U.S.A.). Frozen stocks were stor.ed at -8(PC and reculti~red twice before use. Routinely. mouse ad ren~~ l cells were cultured in 25-cni' plastic tissue cul- ture flasks (Cost;~r, Cambridge, MA, U.S.A.) unless otherwise noted. The adrenal cells were ni;~intnined in minimal essential medium Fl5 (Gibco. GI-and Island, NY, U.S.A.) supplemented with heat-inactivated 10% fetal calf 5eruni (FCS) at 37°C in a humitlified atmosphere of 5% CO, in air according to Llonta (1976). Swine intestinal epitheli;~l cells were collected from fetal swine according to the method of Wilson and Hohmann (1974). using the terminal 25cni of colon. The final suspension of co- lonic epithelial cells wasadjusted tocontnin IOh viable cells/mL. Viability wasdetermined by the exclusion of trypan blue.

Trc.potlc,t?irr k~orI~sc~trtc~t~iI,e strain 878 was originillly isolated from a clinical specimen and has been Ictbo~.atory adapted (Har-

washed with 60mL MB (pH 7.25) to remove unattachecl or- ganisms. The cultul-ed cells were then fixed at 4°C with 2% glutaraldehyde for 18h. The coverslips were removed f~.oni the tubes. twice wi~shed with 30 11iL MB, and clehydl-ated in :igl.aded series of 20-100% ethanol, followed by two changes of 100% amyl acetate. Each sample was dried by Antlel-son's critical point method (I95 I ) , with CO, astransition fluid. The specimens were mounted on ;~lu~ninurn stubs and coated with gold (200 A ( I A = 0. I nni)) ;LI room tempefi~ture with n Kinney KDG-N vacuum evopol-atol. (Kinney Vacuum Co., Boston. MA, U.S.A.). Samples were placed on a tilted rotary pl;~lforni during coating. Each specimen was ex>iniined in an AMK-IOOOA (Acl- vanced Metals Research Corp., Bedford, MA, U.S.A.) scanning electron microscope at 30 kV.

Results Coincubation of T. hyoclv.solto.icre with isolated . .

ris ot rrl. 1972). Stock C U ~ ~ L I I - e s were m>~intained~at -80°C in the swine intestinal epithelial cells or mouse adrenal lyophilized state and, upon reconstitution, were inoculated into 50-mL test tubes containing 30mL trypticase soy broth without

cells in culture, resulted in a direct binding of tre- dextrose (BBL. Baltinlol-e. MD. U,S.A.), The medillm ponemes to the host cell surface. The micl-oscopic supplemented with heat-inactivated 1% FCS and prepared ac- appearance of treponemal attachment is shown in cording to the method of Kinyon and Har.ris (1974). All cultures the light photomicl-ographs (Fig. 1). As indicated, were grown at 37°C in an anaerobic ittmosphere of 70% Hz and tl-eponemes were noted i n val-ious configt,l.ations of 3% CO, for 18h, centrifuged fol- 15 min (12 000 x g) , and thrice washed with supplemented minimal essential medium prior to attachment. Similar results were obtained with coincubation with host cells. isolated swine intestinal epithelial cells.

For coincubation, cultured adrenal cells were challenged at The percentage of parasitized host cells in- 505% confluency to allow for background observ:rtions; isol:~ted swine intestinal epithelial cells were suspended in tissue culture medium to a concentration of 10h viable cellslmL. Both cell types were inoculated with fresh cultures of treponemes ad- justed to 7 x 10' colony-forming units (CFU) per millilitre of tissue culture medium. To maintain host cell viability and mor- phological integrity, incubation was carried out at 37°C in an z~tniosphere of 5% CO, in air. The cultured adrenal cells were then rinsed exhaustively with 300mL Millonig's buffer (MB). pH 7.25, to remove unattached organisms. Swine intestinal epithelial cell - bacterial suspensions were separated by differ- ential centrifugation according to the method of Wilson and Hohmann (1974). Each cell type, containing attached tl-e- ponemes, was then fixed for4 h with 2%glut:u'aldehyde buffered with MB, stained with Giemsa biological stain, and obsel-ved by light microscopy. The bottom of each tissue culture flask was cut out priortoobservation. The percentage of animal cells with three or more attached treponemes was estimated by observing a minimum of 200 adrenal cells in 40 separate fields. Cells with- out added treponemes were included as negative controls ac- cording to Wilson and Hohmann (1974); each cell type was incubated, fixed, and treated under identical conditions to pre- vent observations that might lead to artifact.

The number of viable treponemes was determined by plating serial 10-fold dilutions of each sample on 5% blood agar plates accol-ding to the method of Kinyon et rrl. (1977). One rnillilitre of the initial inoculum 01- a sample from each time interval studied was routinely diluted in sterile tissue culture medium, plated on 5% blood agar plates, and incubated in anaerobic jars with cold palladium catalyst. Each culture dilution plate was examined and the number of CFU counted after 3 days of incubation.

Observations by scanning electron microscopy were mode with cultured adrenal cells grown in 5-cm2 Leighton tubes con- taining a plastic coverslip (9 x 55 mm). The cultured cells were inoculated with 2.0mL of freshly harvested treponemes (7 x lo7 CFUImL) as previously described. After 60min incubation at 37°C in an atmosphere of 5% CO, in air, a large number of spirochetes had attached to the cultured cells. The tubes were

- creased with time post infection. These results are presented in Fig. 2. The percentage of attachment of T. Izyoclyset~tet.iae to swine intestinal epithelial cells is shown for each time interval studied. No significant difference between the mean value of percentage attachment for swine intestinal epithe- lial cells and the mean value of percentage attnch- ment for mouse adrenal cells (not shown) was ob- served when each figure was compared by the Stu- dent's t test. Since the mean values (f SD) for each cell type were essentially identical, it was not prac- tical to plot both figures. The attachment of tre- ponemes began to occur 15 min after infection, and within 60min all cultured cells exhibited attached organisms. When the treponemes were heat- treated at 56°C for 60 min or held at 23"Cfor several days (results in a loss of viability) before coincuba- tion at 3TC, attachment to the cultured adrenal cells or isolated swine intestinal epithelial cell sus- pension was impaired; only rarely could a tre- poneme be found attached to a host cell. A loss in bacterial cell viability, as evidenced by standard plate counts, was not observed throughout the course of inci~bation with host cells (not shown).

Experiments were also designed to determine whether attachment of T. hyodysrtlret~icre was in- fluenced by temperature. When organisms were suspended in tissue culture medium at the temper- ature of incubation and immediately incubated with cultured adrenal cells or isolated swine intestinal epithelial cells, differences in the percentage of at-

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KNOOP ET A L

FIG. 1. Attachment of T. hyoc/y.ret~/er.ir,e to cultured adrenal cells. Various configurations of treponemal attachment to the host cell wel-e observed (large and small arrows). The significance of this finding is not clear (see Discussion). Similar results were observed with isolated swine intestinal epithelial cells. Coincubation was carried out for 60min at 37'C. Photomicro- graphs were taken at a magnification x 1000.

tachment were detected (Table 1). At temperatures studies were carried out with cultul-ed mouse ad- below 37"C, adecrease in the numbel-of parasitized renal cells. Attachment was then observed by host cells was observed. At 42°C the percentage of scanning electron microscopy. The fixation and parasitized host cells was similar to that at 37°C critical point drying techniques preserved the mor- incubation. phological integrity of both the treponemes and

Because of difficulties incurred in culturing cultured cells (Fig. 3). These findings demonstrate swine intestinal epithelial cells, all subsequent several important aspects of the host-bacterial cell

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402 CAN. J . MICROBI

MINUTES AFTER INFECTION

FIG. 7. Percent;rge of isol~rretl swine intestinal epithelial cells pirrasitizetl by nietabolic~iily active T. l~~oc!\..srt~trr.irrp. There was no attachment when the org:rnisnis wel-e heat tre~ited at 56°C for60niin or held ;rt 23°C for3 days. Symbols reprehent percent- age attachment at 37OC to isolatetl swine intestinill epitheli:.; cells for indicated minutes after infec~ion.

TABLE I . Effect of temperature on the attachment of T. /iyorl~~sct~tri~irre to cultured adrenal cells

Tempera t~~re , ' C Percentage attachmeiit"

oCalculared 11s described i n the test after 60 m i n o f coin- cubat ion at 37°C; the s l i ~ n d a r d deviat ion ( + I S D ) i s shown fo r each percentage. S imi lar results were obta ined when the organism was co incubeted w i l h isolated swine intest inal epirhel ial cells a t each respective temperature.

interaction. First, the 01-ganism appeared to attach without observable topogl-aphical alteration of the host cell surface. Second, treponemes were ran- domlv distributed on the host cell: a ~redilection for one specific area of the cultured cells, such as the niicl-ofibl-ils 01. nuclear region, was not detected. Third, the morphology of T. hyo&seilro.ia~ re- mained unaltered except occasionally a straight- ening of the spirals was observed. This may be due to the shrinking of cultured cells during dehydl-a- tion, causing the adherent organisms to stretch lengthwise along their extended axis. Unwound axial fibrils on either end of the treDoneme were often noted in close association with the surface of the host cell (Fig. 3, A and D). The signifi- cance of this observation is not clear.

Discussion Recent research in bacterial attachment and col-

onization of various mammalian cell surfaces has demonstrated the importance of these events in the disease process (Gibbons and van Houte 1975;

OL. VOL. 25. 1979

Savage 1972; Smith 1977). Knowledge of these in- teractions is therefore crucial for an understanding of bacterial pathogenicity. The present studies on the attachment of T . hyocIysc~iltei.icre to isolated swine intestinal epithelial cells or mouse adrenal cells in culture have offered infol-mation on the nature of infection by this organism. Scanning electron micl-oscopic observations on specimens of colonic mucosafrom swine with artificially induced dysentery have depicted spirochetes morphologi- cally compatible with T. hyoc!\~.s~ilfei.inp in large numbers on the epithelial surface (Kennedy et 111. 1973; Kennedy and Strafuss 1976). More I-ecently, Hughes rt (11. (1977) have shown the in vivo in- teraction of T . l~j~ol!\)seilt~~.icr~ with swine intestinal epithelium by immunofluorescence. In the present study, micl-oscopic obsel-vations are supported by quantitative data on the in vitro attachment of T . I~yollysci~tei~icrc. Therefore, the present report pro- vides a model for further studies on the attachment of treponemes at the cellular level. These findings parallel other recent observations on the attach- ment of microorganisnis to mamnialian host cell surfaces (Arbuckle 1972; Beachey and Ofek 1976; Ofek et 01. 1975). GI-oup A streptococci attach to mammalian cells via lipoteichoic acid (Beachey anti Ofek 1976). For gram-negative organisms. cell surface appendages (pili) mediate attachment to host cells (Honda and Yanagawa 1975).

This study was initiated to investigate the in vitro attachment of T. hyot!\veiltei.icre. Since this or- ganism has been shown to attach to swine intestinal epithelium, isolated cells from swine intestine were I-outinely used for comparison with cultured mouse adrenal cells. Although this culture system has been used extensively in the assay of both Vibrio cl~ole~.cre and Escherichia coli enterotoxins (Donta et ( 1 1 . 1973; Donta and Smith 1974; Donta et 01 . 1974; Evan and Wishnow 1974), i t has not previ- ously been used to study bacterial attachment. Cultured adrenal cells, at 50% confluency, could be rigorously observed to assure that no unattached tl-eponenies were adherent in the background or in spaces between cells. This system afforded a rapid, reliable, and I-eproducible method to investigate the attachment of treponemes to host cells by light microscopy. Attachment of the organism to the host cell surface occurred rapidly (within 15 min) and under conditions for maximal attachment (60 min, 37"C), large numbers of organisms adhered to the host cell surface. Fitzgerald et rrl. (1975, 1977), Fieldsteel et 01. (1977). and Hayes er a / . (1977) recently observed that the interaction of pathogenic Trepotzet~i~ palliciut?~ with mammalian

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cells resultecl in attachment; nonpathogenic tre- ponemes failed to attach.

Inaddition to the kineticsofattachment, we were also interested in the manner of treponemal at- tachment to the host cell surfrice. Scanning electron micl-oscopy showed that the organisms attached via their outer surface to the host cell membrane. The background of all samples was completely scanned and no free treponemes were obsel-ved. Several types of treponemnl attachment to the host cell were observed. Althoi~gh the significance of these obsel.vations is not known, other recent st~ldies have demonstrated the polar attachment of T. prflli~11r1,i to human mononucleru- phagocytes (Brause and Roberts 1978).

Relative to the cultured adrenal cells within any given flask, cell vr~riations were obse~.ved. Some cells were elongated (fibroblastic like), whereas others were rounded. In all instances, treponemes attached to both cell types in appl-oximately equiv- alent numbe~-s. In addition, similar numbers of tl-e- ponemes attached to virtually every cell within the flasks. Heat-killed organisms or organisms held at 23°C for sevel-al days failed to attach to the cilltured

Medicine, Minneapolis, MN, U.S.A., foleditol-ial assistance. This investigation was s~~pported by a grant from the National Pork P~.oduce~-s Council.

ANDERSON, T. F. 195 1. Techniq~res forthe preservation ofthree dimensional s tr~~ctur-e in prep;~ring specimens for the electron rnicroscope. Trans. N.Y. Acatl. Sci. 13: 130-133.

ARBUCKL.E. J. B. R. 1972. The att;~chment of Clos/ritlirorr n.c.1- clrii (C. pc,r:fi.irrLgor.s) type C to intestinal villi of pigs. J . Pathoi. 106: 65-72.

BEACHEY, E. H. . and I. OFCK. 1976. Epithelial cell hintling of gl.oup A streptococci by Iipoteichoic acid on fimbriae tlenuded of M protein. J . Esp. Med. 143: 759-77 I.

BRAUSI-., B. D.. and R. B. Roucn-I-s. 1978. Attachment of viru- lent i"r.c,poirr~r~tr ~)ol/itl~rrr~ to human rnor ion~~cle ;~~. phagocytes. Br. J . Vener. Dis. 54: 218-224.

DONTA. S . T. 1976. Interactions of chole~.agenoitl and G,,, ganglioside with ente~.otoxins of Vibrio c~lrol(,r.oe antl E.s- c~lreric.lrio c,oli in c u l t ~ ~ r e t l ntlrenal cells. J . Infect. I>is. 133: 115-119.

DON-[.A, S. T., M. K I N G . and K. SI-OPER. 1973. Induction of steroidogenesis in tissue culturc by cholera enterotoxin. NLI- t~ i re (London). 243: 2461-47.

DoNT,\, S. T., H. W. MOON. i~nd S . C. WHIPI'. 1974. Detection of hea-labile E.sclrc~ric~hitr c,oli enterotosin with the use of adrenal cells in tissue culture. Science. 183: 334-336.

DONI.A, S. T., antl D. M. S ~ I I - I . H . 1974. Stimulation of teroi t l - ogenesis in tissue culture by enterotoxigenic E.sc~lrr~r~ic~lrio coli and its neu t~~l iza t ion by specific a n t i s e r ~ ~ m . Infect. Immun. 9:

cells. This observation h;is also been noted for the jw-jo5. of T. pLll l j l~l l l , l to CUltllred cel lS DUNNI;. H. W.. and A. D. LIJXIAN. 1975. DiseaseoFswine. 4th

edn. The Iowa State Univel-hity Prehs. Ames, IA. p. 541. (Fitzger.ald ct (11. 1977). The inability of T. hqo~ly- . EV,\N. G, N,, and M , WISHNOW, 1971, Esc.llO,.ic.llio selitel.iLrc or T . p f l l l i ~ / / / /?? to attach after heat treat- en te ro toxin- indu~l steroitlogenesis in cultured atlrenal merit or 'clgilig' may suggest the presence of tumor-cells. Infect. Immun. 10: 146151.

thermolablle or metabolically act ive surfiice re- F I I ~ L D S I E E L , A. H. . F . A. BECKER. and J . G. S iou- r . 1977. Prolongetl survival of virulent Treporrrr~ro ptrllitlrrr~r (Nichols ceptor. strain) in cell-free and tissue culture systems. Infect. Immun.

Other recent studies have correlated the in vitl-o 18: 173-182. attachment of microol-ganisms with virulence. The FITZGERALD. T. J . . R . C. JOHNSON. J . N. MILLER. k~nti J . A. ability of virulent ~ h i ~ - e l l a sp. and certain entero- pathogenic Esc~heric17ia col i to induce disease is related to their ability to attach and infect cultured cells (LaBrec et 01. 1964: Ogawa (11. 1967; Osada er 01. 1972; Watkins 1960). Swanson (1973), using I-adiolabeled microorganisms, has shown that vir- dent Neisseriu go~z(>~.rIioerr(~ (colony type 2) attach to cultured r~niniotic cells, whereas avirulent cells (colony type 4) attach less readily.

These observations concerning tl-eponemal at- tachment provide a possible model for studies on the pathogenesis of T. hyorlysenrerirrc at the cellu- lar level, for elucidation of host and parasite re- ceptors, and for testing possible effective prophy- lactic vaccines and pharmacologic agents.

Acknowledgnlents

The authors thank J . M. Kinyon and D. L. H x - ris, College of Veterinary Medicine, Ames, IA, U.S.A., for graciously supplying cultures of Tre- po~ze~?ltr IlyocIysc~ztc~.ic~e (strain B78) and T. J . Fitzgel-ald, University of Minnesota School of

SYKES. 1977. Characterization of the attachment of 7'r.c.- 17oire11ro p(~llitlrrrrr (Nichols strain) t o cultured rn:~nimnlii~n cells and the potential reliitionship of attachment t o patho- genicity. Infect. Immun. 18: 467-478.

F1.r-ZGERALI), T. J . , J . N. MILLER. and J . A . SWES. 1975. i"rc,poirc,rrro ptrllitlrrr~r (Nichols stl-ain) in tissue cultures: cel- lula~. attachment, entry and surviv:~l. Infect. Imnlun. 11: 1 133- 1140.

GIBBONS. R. J . . antl J . A. V A N H 0 u . r ~ . 1975. Bacterial adher- ence in 01-21 microbial ecology. Annu. Rev. Microbial. 29: 19-14.

HARRIS. D. L.. R. D. CLOCK. C. R . CHRIS.I .ENSEN. and J . M. KINYON. 1972. Swine dysentery: 1. Inocul:~tion of pigs with Trepor~c,r~rtr Il~ocl~.sc~rr/c~~.ioc~ (new species) and reproduction of the tlisease. Vet. Med. Small Anim. Clin. 67: 61-63.

HAYES. N. S., K. E. MUSE, K. M. C O L L I E R , antl J . B. BASE- MAN. 1977. Palnsitism by virulent T).eporror~tr ptrllitl~rr~~ of host cell surfaces. Infect. Immun. 17: 174-186.

HONLIA. E.. and R. YANAGAWA. 1975. Attachment of Cory~rr- htrc./eriirrrr rc,rrcilc, t o tissue culture cells by the pili. Am. J . Vet. Res. 36: 1663- 1666.

HUGHES, R.. H . J . OLANDER. D. L. KANI-I -Z . and S. Q U R E S H I . 1977. A study ofswine dysentery by immunofluol.escence and histology. Vet. Piit hol. 14: 390-407.

K E N N E D Y , G. A,. ilntl A . C. S ' r n ~ ~ u s s . 1976. Scanning electron niicro.scopy of the lesions of swine dysentery. Am. J . Vet. Res. 37: 395-401.

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