MICROBIOLOGY LEll-ERS

ELSEVIER FEMS Microbiology Letters 132 ( 1995) 247-25 I

Pili of Pasteurella multocida of porcine origin

Richard E. Isaacson ‘.*, Emilio Trigo ’

Received 15 August 1995; accepted I6 August 1995

Abstract

Using electron microscopy, pili with at least two distinct morphologies were observed on strains of Pusteurda

mufrocidu isolated from pigs with atrophic rhinitis. Rigid pili were found on 60%80% of all cells observed. These pili had a strong tendency to lie flat along the side of the outer cell membrane of P. multocidu and as a result frequently were difficult to see. After growth in vitro, piliated P. mulfocidu cells produced few pili (approx. 3-5 per cell). Heavily piliated cells were occasionally observed. The second type of pili were curly and also were difficult to visualize. Cells from cultures containing piliated cells failed to attach to red blood cells and to immobilized mucus.

Kr~~ord.~: Postrurdltr multocidu: Pili; Adhesion: Atrophic rhinitih

1. Introduction

Atrophic rhinitis is an infectious disease of the upper respiratory tract of swine that is the result of a

dual infection with both Pasteurella multocida and Bordetella bronchiseptica [ 1,2]. Clinically, atrophic

rhinitis is associated with mild to severe atrophy of nasal turbinates. P. multocida produces a potent dermonecrotic toxin (DNT) which is the only known

virulence factor that functions in this disease [3].

When administered to pigs, purified DNT is capable of eliciting all of the clinical signs associated with atrophic rhinitis including sneezing, nasal discharge,

the twisting of the snout, and atrophy of nasal

’ Corresponding author. Tel.: + I (217) 333 7825; Fax: + I (2 17) 333 4628: E-mail: iaaacson@uxl .cso.uiuc.edu.

turbinates [4]. Therefore, it has been concluded that

P. multocida is the principal agent that causes at-

rophic rhinitis. However, while the cell-free toxin can replicate the disease, direct intranasal instillation of toxigenic P. multocida does not result in the

colonization of this site nor in disease [S-8]. To experimentally induce clinical disease, pigs must be

pre-exposed to B. bronchiseptica. When followed with toxigenic P. multocida, B. bronchiseptica facil-

itates the colonization of the upper respiratory tract

by P. multocida. The requirement of B. bronchisep-

tica can be obviated in two ways. The first is to

expose the nasal mucosa of the pig to dilute acetic acid [9] and the second is to expose the nasal mucosa to cell-free filtrates of media used to culture B.

bronchiseptica [7]. B. bronchiseptica also produces a dermonecrotic toxin and cell-free filtrates of culture media contain this toxin[ IO]. Unlike the P. multo-

0378.1097/95/$09.50 0 1995 Federation of European Microbiological Societies. All rights reserved

SSDl 037% IO97(9.5)003 17-7

248 R.E. Isaacson, E. Trigo/FEMS Microbiology Lettrn 132 (IYYSI 247-251

cidu DNT, the DNT of B. bmnchiseptica is not

potent and does not induce clinical atrophic rhinitis.

Therefore, it has been concluded that the requirement of B. bronchiseptica is to aid in the colonization of

the nasal mucosa by P. multocida. The toxin of B.

bronchiseptica probably causes minor tissue damage to the nasal mucosa and this damage permits P.

multocida to colonize the damaged sites.

The colonization of mucosal surfaces is frequently

mediated by adhesive structures called pili [l I]. Pili

that are adhesive promote colonization of mucosal

surfaces by spanning the distance between the bac-

terium and a specific receptor on the host tissue.

Through this process, the attached organisms are

able to resist the natural host mechanisms that pro-

mote clearance of the organisms from that site. Attachment leads to colonization. One hypothesis regarding the function of the B. bronchiseptica toxin

is that it exposes P. multocida-specific receptors

through the alteration of target surfaces components, possibly by damage to the tissue. If this hypothesis is

correct, P. multocida must produce adhesins and these adhesins may be pili. Several studies have been

published describing the presence of pili on P. mul-

tocidu [ 12-141. In these reports it has generally been

shown that P. multocida strains that cause atrophic

rhinitis do not produce pili but do attach to porcine nasal mucus. Glorioso et al. [ 141 showed that, while

many P. multocidu strains were adhesive and pro- duced pili, strains that cause atrophic rhinitis did not.

In a preliminary report, Trigo and Pijoan [13] re- ported the presence of pili on toxigenic P. multocidu

isolated from swine. However, the number of pili found on those isolates were quite low and most cells did not have any pili. In that study there was no

attempt to determine the percentage of cells produc- ing pili or to determine if cells from cultures contain-

ing piliated cells were adhesive. In an attempt to

assess adhesiveness of P. multocida strains associ- ated with atrophic rhinitis, Letellier et al. [ 151 devel- oped an adhesion assay using porcine nasal mucus. In those studies, P. multocida strains were shown to be adhesive. In this manuscript we demonstrate that toxigenic strains of P. multocida that cause atrophic rhinitis reproducibly produce pili, that they can pro- duce at least two morphologically distinct pili, and that these strains are not adhesive in two established in vitro binding assays.

2. Materials and methods

2. I. Strains and mediu

Toxigenic P. multocidu strains D7 and D9 were

isolated from pigs with atrophic rhinitis. Strain D7 produced a D capsule and strain D9 produced an A

capsule. Cells were grown at 37°C in lactose broth

(0.3% beef extract, 0.5% peptone, 0.5% lactose, and

10% pig serum>. A B. bronchiseptica strain obtained

from a pig with atrophic rhinitis was grown in

lactose broth or trypticase soy broth (Difco, Detroit,

MI).

2.2. Electron microscopy

Cells were collected after growth by centrifuga-

tion and gently resuspended in saline. Copper grids coated with formvar and carbon were floated on a

drop of resuspended culture for 1 min, removed from the drop, and then allowed to air dry. The grids were

then floated on a drop of sodium phosphotungstate (1%). After 1 min, excess liquid was removed by

touching the edge of the grids with a piece of filter

paper. The grids were then allowed to air dry. Sam-

ples were observed in a Joel transmission electron

microscope.

2.3. Binding assays

Citrated blood (rabbit, pig, and human group 0) was collected and used for the microhemagglutina-

tion as previously described [16]. A mucus binding assay was adapted from the

procedure of Letellier et al. [15]. Mucus was col-

lected from pigs by washing the nasal passages with phosphate-buffered (0.05 M, pH 7.2) saline (PBS).

The mucus was clarified by centrifugation at 1500 X g for 15 min and the concentration of protein in the

wash was determined using the Micro BCA kit (Pierce Chemical Co., Rockford, IL). 100 pg of mucus protein in a volume of 100 ~1 was added to the wells of microtiter plates and incubated overnight at 37°C. The wells were washed with PBS. P.

multocida cells were added to the wells and incu- bated at 37°C for 30- 120 min. Free cells were gently removed by three successive washes with PBS. Ad- herent P. multocida were removed from the plates

R. E. Isaacson. E. Trip) / FEMS Microhiolog~ Lerrerv 132 C 1 YY.5) 247-251 249

by the addition of Triton X-100 (0.5%) and the

number of P. mulrocidu in the Triton X-100 wash was determined by serial dilution and plating onto

sheep blood agar plates. This concentration of Triton X-100 did not affect the viability of P. multocidu.

3. Results

3.1. Pili of P. multocidu

In a preliminary study, various media were em-

ployed to determine the best growth medium for pilus production. Media tested included trypticase

soy broth, trypticase soy agar, blood agar, chocolate

agar, MEM + glucose, tryptone yeast broth, Luria-

Bertani broth, and lactose broth. Lactose broth was selected for use because it consistently resulted in

the highest concentration of piliated cells. Both

strains of P. multocidu (D7 and D9), when grown in lactose broth, produced pili. Between 60 and 80% of

cells had 3-5 pili per cell (Fig. I). Generally, the pili

were rigid with an average diameter of approxi- mately 7-10 nm. Morphologically, these pili were

similar to type I pili of Escherichiu coli [ 171. On many cells, the pili were found lying close to the

cell’s outer membrane. When this occurred, the pili were very difficult to visualize. Occasionally, cells

were found that were heavily piliated (Fig. 2). These pili also were rigid with an average diameter of approximately 10 nm. However, these pili were much

longer than those on the poorly piliated cells. Whether the longer pili are different from the pili on the

lightly piliated cells cannot be determined with the

available data, although they also appear to be slightly thicker. The pili on the heavily piliated cells had a

strong tendency to aggregate, forming bundles simi- lar to type I pili of E. co/i [ 171..

On some cells, a fuzzy, poorly defined structure was observed on the outer surface of the outer membrane. These structures were difficult to resolve. When cells were stained optimally, these structures were seen as curly filaments (Fig. 3). These struc- tures were similar in morphology to the pili desig- nated curli from E. coli [ 181. Because these struc- tures were difficult to observe, the relative fraction of cells possessing the ‘curli’ pili was not deter- mined. The overall impression was that many cells

Fio ti’ I. Electron micrograph of P. nudrocidu cells stained with

sodium phosphotungstate. Pili can be seen on the surface of these

cells. The&e pili had diameters between 7 and IO nm.

(approx. 50%) possessed fuzzy surface structures and

that these structures could represent the ‘curli’ pili.

A preliminary examination of some of the condi- tions that supported pilus production demonstrated

that pig serum was not required for piliation. When

serum was left out of the growth medium, the same fraction of cells produced pili. Pilus production also

was not affected by incubation for 24 or 48 h and by incubation at 32°C or 37°C.

Since B. bronchisepticu is important in predispos- ing to colonization, experiments were performed to determine if this organism may exert its action by enhancing pilus production by P. multocidu. B.

bronchisepticu cells were cultured in either lactose broth or trypticase soy broth for 24 h. The cells were removed by centrifugation ( 10 000 rpm for 20 min). The clarified supematants were then added to fresh lactose broth (I: 10, v/v) and inoculated with P.

250 R.E. l.wuc.sor~, E. Tri~o / FEMS Microhiolo~y Lrttri-.s 132 f lYY.51 247-251

Fig. 2. Electron micrograph of a heavily piliated P. multocidu cell

stained with sodium phosphotungstate. These pili had diameters of

approximately 10 nm.

multocidu strain D7 or D9. After growth, the cells

were examined for pili. No enhancement or suppres-

sion of pilus production was observed.

Fig. 3. Electron micrograph of a piliated P. nudtncidu cell stained

with sodium phosphotungstate. This cell contains numerous pili

that appear ‘curli’.

3.2. Binding as.sa_vs

Previously it was shown that P. multocida strains

of porcine origin attached to human group 0 red

blood cells [14,19] and to mucus obtained from

porcine respiratory tract [IS]. Other experiments

demonstrated that P. multocidu of non-porcine ori-

gin attached to squamous epithelial cells from the pharynx of rabbits and to HeLa cells [14]. Because

we were interested in assessing the role that the pili

on P. multocidu played in adhesion and colonization of the nasal cavity, hemagglutination and mucus

binding assays were performed with P. multocidu

strains D7 and D9. Neither strain hemagglutinated

rabbit, pig, or human group 0 erythrocytes despite the fact that they produced pili. However, two P.

multocidu strains isolated from rabbits (producing A

or D capsules) did hemagglutinate the human group

0 erythrocytes. Unlike the strains employed by Letellier et al. [15], these P. multocidu strains also

did not bind to immobilized porcine nasal mucus and neither did the two isolates from rabbits.

4. Discussion

The results presented demonstrate that toxigenic

strains of P. multocida associated with atrophic

rhinitis indeed do produce pili. This confirms the previous work of Trigo and Pijoan [ 131, who also

observed pili on P. multocida. The results reported

here differ from those of Trigo and Pijoan [ 131 in

that pili were found on a large number of cells and heavily piliated cells were observed. Furthermore,

the P. multocidu strains used produced pili with at least two distinct morphologies. The identification of

the ‘curli’ pili on P. multocidu is a new observation. The rigid pili were found on 60-80’S of all cells observed. However, since these pili had a strong

tendency to lie flat along the side of the outer cell membrane of P. multocidu, their presence was fre- quently obscured. Unlike most enteric bacterial pathogens, after growth in vitro, P. multocidu cells produced few pili (approx. 3-5 per cell). The mor- phology and the apparent distribution of the rigid pili is similar to what was seen by Trigo and Pijoan [ 131. At this time it is not clear if the heavily piliated cells represent quantitative variants in pilus production or

R. E. lsaucsot~. E. Trigo / FEMS Microhiolo~~ Lurtrrs 132 (19951 247-251 251

whether the pili on the heavily piliated cells may

represent a third class of pili. Consistent with the

latter, pili on heavily piliated cells were longer and had a larger average diameter than pili on poorly

piliated cells. The discovery of the ‘curli’ pili was not antici-

pated, The curli pili were similar in morphology to curli found on E. coli [ 181. Curli have been estab-

lished as important adhesins on these enteric

pathogens and are believed to recognize fibronectin

as a receptor. The role of the curli pili on P. multocidu in atrophic rhinitis is unknown, but by

analogy to the enteric pathogens may serve as an

adhesin that possibly recognizes fibronectin. The

previous observations that either dilute acetic acid [9] or B. hmnchisq~ticu toxin [20] predisposes the nasal

cavity to colonization by P. multocida leads to the

hypothesis that the functional receptor for P. multo-

cidn binding is not normally exposed on the nasal

mucosa surface. Since both the treatment with dilute

acetic acid or with B. hnchisepticu toxin should

result in tissue damage to the nasal mucosa, it would be predicted that a substance that could serve as a

receptor could be uncovered through this destructive

process. It would be anticipated that included among those substances uncovered through this process

would be fibronectin. However, whether the ‘curli’

pili or any of the rigid pili function as an adhesin in

vivo and whether they recognize fibronectin is un- known and warrants further investigation.

It was somewhat surprising that neither P. mu/to-

cidu strain from pigs attached to erythrocytes or to

porcine nasal mucus even though more than half of

the cells produced pili. These results lead to several interpretations: (i) none of the pili observed by elec- tron microscopy were adhesins; (ii) the pili were

adhesins but were not present in sufficient numbers after growth in vitro to effectively mediate attach-

ment; (iii) the receptor was not accessible to the P. multocidu cells in either assay; (iv) colonization of

the nasal mucosa is mediated by a process that does not involve adhesion; (v) P. multocidu do not attach

to the erythrocytes or mucus but do attach to tissue specific receptors that were not present in the assay mixtures. While it is not possible to distinguish between these possibilities. we favor interpretation nos. (iii) and (v). As described above, tissue damage

appears to be a necessary prerequisite for P. muito-

cidu to colonize nasal mucosa of pigs. Since no

effort was made to alter the erythrocytes or mucus, it

is likely that the receptor was not exposed and, therefore, not available to bind the adhesive pili. Further studies to clarify these possibilities are in

progress.

Acknowledgements

The technical assistance of Stephen Deisher and

LuAnn Miller are acknowledged.

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