Ornithobacterium rhinotracheale (ORT) is a pleomorphic,
rod-shaped, gram-negative bacterium associated with respiratory disease
in poultry. The poultry industry has suffered significant financial losses
because of the drop in egg production, growth suppression, mortality and
condemnation of carcasses in flocks infected with this organism (Van Empel
and Hafez, 1999; Lopes et al., 2000; Hafez, 2002; Chansiripornchai,
2004). The bacterium primarily infects the trachea, lungs and air sacs,
but can also manifest as a systemic disease with hepatitis, joint lesions
and cerebrovascular pathology (Van Empel et al., 1997; Van den
Bosch, 1998; Schuijffel et al., 2005). Concomitant infections with
other respiratory pathogens appear to aggravate the symptoms but are not
required for causing disease (Schuijffel et al., 2005). At this
time, up to 18 different serotypes of ORT have been identified (Van den
Bosch., 1998; Schuijffel et al., 2005). Serotype A represents 94%
of all isolates from chickens, whereas it represents 57% of isolates from
turkeys. Serotype D and F have not been isolated from chickens, nor has
Serotype G been isolated from turkey (Joubert et al., 1999).
O. rhinotracheale control can not be achieved effectively through
antibiotic use. Results from antibiotic sensitivity tests of 45 strains,
collected at 45 different broiler farms with clinical respiratory disease,
has demonstrated that acquired resistance against commonly used antibiotics
is very high (Cauwerts et al., 2002).
The mechanisms via which ORT causes disease are largely unknown. Pathology
on infected animals suggests that the bacteria colonize the respiratory
tract of the host by adhering to host mucosa. Treatment of the disease
is complicated, as most isolates of ORT are resistant to most commonly
used antibiotics. Vaccination is an effective form of infection prevention.
Available vaccines composed of killed ORT however, are assumed to give
only partial protection against a limited number of strains (Van den Bosch,
To understand the role of outer membrane proteins (OMPs) in the pathogenesis
of ORT and also to contribute in developing novel strategies for immunization
of chickens against the bacteria, we studied the effect of polyclonal
anti-OMPs sera in bacterial adherence inhibition assays.
MATERIALS AND METHODS
Bacterial cultures: An ORT serotype A, isolated from commercial
chickens was used. Bacteria were grown onto Columbia agar (Lab M, UK)
for 48 h at 37°C in 5% CO2. Bacterial colonies were collected,
washed three times with phosphate-buffered Saline (PBS), pH 7.4 and finally
resuspended in PBS to yield an optical density of 0.6 at 550 nm. The suspension
contained 3-3.5x108 colony-forming units of ORT per mL and
was used for immunization and adherence studies.
Extraction of OMPs: For extraction of OMPs, ORT was first grown (48 h, 37°C, 5%
CO2) onto Columbia agar and then in 200 mL of Todd-Hewitt broth
(Biomark, India) for 48 h at 37°C, with shaking at 150 rpm. Bacteria
were collected by centrifugation (4000xg, 15 min, 4°C) and resuspended
in 20 mL of 10 mM Tris-HCl, pH 7.4. OMPs were extracted as described by
Lopes et al. (2000) and Chansiripornchai (2004) and analyzed by
Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis (SDS-PAGE),
using a 4% stacking gel and a 10% separating gel.
Purification of major OMPs from acrylamide gel: Extracted OMPs were electrophoresed in a 10% gel and visualized with 0.25 M KCl. Three major OMPs of 45, 53 and 70 kDa were
cut from the gel and placed in separate dialysis tubes, containing 1.5
mL SDS-PAGE running buffer. The tubes were electrophoresed in a horizontal
chamber for 30 min and dialyzed against PBS, overnight at 4°C. After
dialysis, the gel pieces were discarded and tubes contents were concentrated
with Nitrogen gas. The purified OMPs were analyzed by SDS-PAGE and quantified
by dye-binding method.
Chickens: Day old commercial broiler chickens were procured from a breeder
farm and grown until they became specific antibody negative (SAN). SAN
chickens were used for production of antisera and also for preparing tracheal
tissues for bacterial adherence inhibition assays. To ensure the chickens
were free of maternal antibodies, they were tested by a commercial ORT-ELISA
kit (IDDEX, USA) at days 1, 14 and 28.
Antisera: Antisera were prepared in SAN chickens. Total extracted OMPs or
purified 45, 53 and 70 kDa OMPs were emulsified in ISA70 oil adjuvant
at the ratio of 3 to 7 and injected subcutaneously to 4 weeks old SAN
chickens (30 μg in a final volume of 0.5 mL/chicken). One more injection
was given two weeks after the first immunization and the chickens were
bled two weeks later. Sera were stored at -20°C until use. Antiserum
against the whole cells of ORT was prepared as above, after inactivating
the bacterial suspension with 3% of formalin at 60°C for 30 min.
Immunodot and Immunoblotting assays: In immunodot assay, antigen spots were made on small pieces of nitrocellulose
membrane, using 10 μL volumes of the extracted total OMPs. The membranes
were blocked in 5% skim milk in PBS, washed three times in PBS containing
0.05% Tween (PBS-T) and probed with 1/100 dilutions of sera. After washing
as above, the membranes were incubated in a 1/200 dilution of a peroxidase
conjugated goat anti-chicken IgG (KPL, USA) in PBS-T. The membranes were
washed again and developed by 4-chloro-1-naphtol (Sigma, USA).
For immunoblotting, the extracted total OMPs were electrophoresed in
a 10% gel and transferred to Nitrocellulose membrane. The membrane was
cut in strips, blocked with skim milk and probed with the sera and conjugate
Adherence studies: Adherence studies were performed as described by Gyimah and Panigraphy
(1988), with some modifications. Two hundreds microliter volumes of the
ORT suspension were incubated with 200 μL of each antiserum for 1
h at room temperature with occasional shaking. As a control, 200 μL
of ORT suspension was incubated with 200 μL of a normal chicken serum.
Each of the treated bacterial suspensions was added to 5 tracheal sections
(1x3 mm rectangular sections), maintained in 4 mL of Krebs Ringer Tris
saline (NaCl 7.5 g L-1, CaCl2 0.305 g L-1,
MgSO4.7H2O 0.318 g L-1, KCl 0.383 g L-1,
buffered with 0.05M Tris-HCl), pH 7.4, in a 50 mL conical tube. Bacteria
and tissue sections were incubated for 1 h at 37°C in a shaker. After
incubation, the tracheal sections were washed in three changes of PBS
to dislodge non-adherent bacteria. Each tracheal section was ground in
2 mL of PBS using a sterile mortar and pestle and subjected to bacterial
counting following two fold serial dilutions and plating on Columbia agar
medium. Relative adherences were determined as follows and compared by
OMPs of an ORT serotype A were extracted and analyzed by SDS-PAGE and silver
staining. The OMPs bands were in range of 32 to 83 kDa. Three major OMPs with
apparent molecular weights of 45, 53 and 70 kDa were purified from the gel by
electro elution (Fig. 1).
Antisera to ORT, total OMPs and each of the three eluted outer membrane
proteins were prepared by subcutaneous injections of commercial broiler
chickens. Two weeks after the second injection, the presence of relevant
antibodies in the immune sera was demonstrated by immunodot but the control
chickens and chickens immunized by 45 kDa OMP had not any ORT-reactive
antibodies (data not shown).
analysis of OMPs of ORT. Lane 1 to 6 represent the
molecular weight markers, whole cell of ORT Serotype A, OMPs extracted from
ORT, purified 70 kDa OMP, purified 45 kDa OMP and purified 53 kDa OMP respectively.
The proteins were visualized by silver staining
Reactivity of antisera prepared against OMPs of ORT in immunoblotting.
Lanes 1 to 4 represent the reactivity of sera against OMPs extracted from
ORT, purified 70 kDa OMP, purified 45 kDa OMP and purified 53 kDa OMP respectively.
Lane 5 indicates the reaction of normal chicken serum. Molecular weights
are indicated at left
The specificity of antigen-antibody reactions was confirmed by immunoblotting
(Fig. 2). The immune sera against total OMPs, 53 and
70 kDa OMPs were capable of detecting respective antigens, but sera of
control chickens and chickens immunized by 45 kDa OMP couldn't detect
the OMPs antigens.
Inhibitory effects of antisera against OMPs of ORT on the
adherence of ORT Serotype A to chicken tracheal epithelium. Inhibitory effects
of normal chicken serum (control) and antisera against OMPs of ORT, whole
cell of ORT and purified 53 and 70 kDa OMPs have been indicated from left
to right, respectively
The ability of immune sera and control serum to inhibit adherence of
ORT to tracheal epithelial cells, has been shown in Fig 3.
Serum against the 53 kDa OMP significantly (p<=0.05) showed the highest
inhibition of adherence (relative adherence of 22 and 78% adherence inhibition).
Sera prepared against the whole cell antigen of ORT, total OMPs and 70
kDa OMP inhibited the adherence of ORT by 44% (relative adherence of 56%),
34% (relative adherence of 66%) and 20% (relative adherence of 80%), respectively.
Adhesion of bacterial pathogens to epithelial cells is a key step
in the establishment of most mucosal infections and thus an attractive
target of infection intervention and prevention. Chansiripornchai (2004) has shown that lipopolysaccharide (LPS) of ORT inhibited bacterial adherence
to human epithelial cell up to 90%, but the adherence of ORT to chicken
epithelial tissues was not studied. On the other hand, OMPs of ORT may
play a role in receptor (s) recognition, because the role of OMP (s) in
adherence has been shown for many of bacteria like Heamophilus ducreyi
(Dinitra et al., 2005), Actinobacillus pleuropneumoniae
(Enriquez-Verdugo et al., 2004) and Haemophilus influenzae
(Liu et al., 2004).
In the present work, we studied the probable role of ORT OMPs in adherence
to chicken tracheal epithelium, based on the abilities of anti-OMPs antibodies
to inhibit the bacterial adherence. The results of this study showed that
antibodies raised against an OMP of 53 kDa could inhibit bacterial adherence
up to 78%. Inhibitory effect of anti 53 kDa antibodies was significantly
higher than those of antibodies produced against total OMPs and whole
cell bacteria. The difference could be related to the amount of 53 kDa
antigen in these preparations or the effect of other bacterial component
(s) which might affect the immunogenicity of 53 kDa OMP. As we showed
(Fig. 2 lane 1), the 53 kDa OMP was not detectable by
antibodies prepared against the total OMPs.
Considering the results of this study, it can be concluded that OMPs
of ORT could be implicated in bacterial adherence. Although Chansiripornchai
(2004) hasn't used chicken trachea in his adherence studies, synchronous
role of OMP (s) and LPS of ORT in attachment can be envisaged. In fact,
ORT may recognize different receptors and bacterial OMP(s) and LPS may
be implicated in the interaction with different kinds of receptors.
So far there have been a few studies on the OMPs of ORT. Lopes et
al. (2000) investigated the use of OMPs of ORT in enzyme-linked immunosorbent
assay (ELISA) to detect the exposure to ORT infection. They showed that
OMPs-based ELISA was able to detect the exposure to ORT better than the
agglutination test. Recently, in order to develop a cross protective subunit
vaccine against ORT, Schuijffel et al. (2006) showed that immunization
of chickens with a recombinant OMP of 32.9 kDa could reduce the respiratory
pathology of ORT up to 74.6%. This OMP had been identified by screening
of an ORT expression library with sera of live-vaccinated birds.
Based on the results we presented in this research, 53 kDa OMP could
be a candidate for further studies in order to develop a recombinant vaccine