A comparative study of avian bordetella-like strains, Bordetella bronchiseptica, Alcaligenes faecalis and other related nonfermentable bacteria


Hinz, K.H.; Gluinder, G.; Romer, K.J.

Avian Pathology 12(2): 263-276

1983


Cultural and biochemical characteristics, electrophoretic patterns of soluble proteins, and pathogenicity in turkey poults, of 30 avian gram-negative nonfermentable bacterial strains acquired from various geographical regions, of which 26 had been isolated from turkeys and one each from a chicken, duck, goose and sharp-tailed munia, were compared to those of Bordetella bronchiseptica, Alcaligenes faecalis, Alcaligenes denitrificans, Achromobacter xylosoxidans and Bordetella parapertussis. With the exception of two turkey isolates, the avian nonfermentable strains constituted a very homogeneous taxon with characteristics of the turkey coryza agent, which has been tentatively designated as a Bordetella-like bacterium. On the basis of the features studied it could be clearly differentiated from B. bronchiseptica, A. faecalis and the other bacteria, suggesting that the Bordetella-like bacterium occupies a distinct taxonomic position. No relevant differences were found between the strains of turkey coryza isolated in Germany and those from USA. The remaining two bacterial strains from turkeys showed the typical characteristics of B. bronchiseptica and A. faecalis respectively.

Avian
Pathology,
12:
263
276,
1983
A
COMPARATIVE
STUDY
OF
AVIAN
BORDETELLA-LIKE
STRAINS,
BORDETELLA
BRONCHISEPTICA,
ALCALIGEN'S
FAECALIS
AND
OTHER
RELATED
NONFERMENTABLE
BACTERIA'
K.
-H.
H1NZ,
G.
GLUNDER
and
K.J.
ROMER
Klinik
fur
Gefliigel
der
Tierorztlichen
Hochschule
Hannover
D-3000
Hannover
1,
Bischofsholer
Damm
15
Bundesrepublik
Deutschland
SUMMARY
Cultural
and
biochemical
characteristics,
electrophoretic
patterns
of
soluble
proteins,
and
pathogenicity
in
turkey
poults,
of
30
avian
gram
-
negative
nonfermentable
bacterial
strains
acquired
from
various
geo-
graphical
regions,
of
which
26
had
been
isolated
from
turkeys
and
one
each
from
a
chicken,
duck,
goose
and
sharp
-tailed
munia,
were
com-
pared
to
those
of
Bordetella
bronchiseptica,
Alcaligenes
faecalis,
Alcaligenes
denitrificans,
Achromobacter
xylosoxidans
and
Bordetella
parapertussis.
With
the
exception
of
two
turkey
isolates,
the
avian
nonfermentable
strains
constituted
a
very
homogeneous
taxon
with
characteristics
of
the
turkey
coryza
agent,
which
has
been
tentatively
designated
as
a
Bordetella-like
bacterium.
On
the
basis
of
the
features
studied
it
could
be
clearly
differen-
tiated
from
B.
bronchiseptica,
A.
faecalis
and
the
other
bacteria,
suggesting
that
the
Bordetella-like
bacterium
occupies
a
distinct
taxonomic
position.
No
relevant
differences
were
found
between
the
strains
of
turkey
coryza
isolated
in
Germany
and
those
from
USA.
The
remaining
two
bacterial
strains
from
turkeys
showed
the
typical
characteristics
of
B.
bronchiseptica
and
A.
faecalis
respectively.
INTRODUCTION
Since
animal
and
economic
losses
from
enzootic
and
epizootic
respiratory
diseases
in
turkeys
continue
to
increase,
research
teams
have
concentrated
on
determination
of
the
aetiology
and
methods
of
control
of
such
conditions.
The
situation
has
clarified
over
the
last
few
years,
but
there
is
still
considerable
confusion
over
various
agents
associated
with
the
turkey
coryza
complex.
The
clinical
terms
turkey
coryza,
rhino-
tracheitis
or
acute
respiratory
disease
that
are
used
to
describe
the
diseases
do
not
help
to
clarify
the
aetiology,
because
similar
clinical
and
gross
pathological
alterations
may
be
produced
by
different
infectious
agents
and
their
combinations.
Received
27
September
1982
Accepted
24
November
1982
1
Parts
of
these
results
were
presented
at
the
VIIth
International
Congress
of
World
Veterinary
Poultry
Association,
Oslo,
Norway,
1981.
264
K.
-H.
Hinz
et
al.
One
of
the
infectious
agents
that
is
able
to
produce
a
respiratory
disease
in
turkey
poults
has
now
been
identified
as
a
gram
-negative
motile
obligatory
aerobic
nonfer-
mentative
and
nonsaccharolytic
bacterium
(Hinz
et
aL
,
1978;
Simmons
et
al.,
1979).
This
bacterial
agent
was
preliminarily
designated
as
a
Bordetella-like
(BI)
bacterium
(Hinz
et
aL
,
1979a).
Based
on
physical
and
biochemical
properties,
Simmons
et
al.
(1980)
classified
it
as
Alcaligenes
(A)
faecalis.
The
purpose
of
this
study
was
to
provide
differential
characteristics
of
the
organism
as
an
approach
to
its
identification
and
classification,
with
special
reference
to
Bordetella
(B)
bronchiseptica,
A.
faecalis
and
other
related
bacteria.
MATERIAL
AND
METHODS
Bacterial
strains
The
designation
and
sources
of
the
strains
used
in
this
study
are
listed
in
Tables
1
and
2.
Strains
were
checked
for
purity
and
stored
in
the
freeze-dried
state
until
used.
Working
cultures
were
maintained
as
suspensions
in
steamed
milk
at
-70
°
C.
When
a
test
series
was
initiated,
frozen
cultures
were
thawed
and
one
passage
on
blood
agar
(containing
Columbia
agar
base
Oxoid
and
7%
defibrinated
ox
blood)
or
veal
infusion
agar
(Difco)
was
made
before
use.
With
the
exception
of
one
isolate,
all
the
avian
strains
have
been
isolated
from
the
respiratory
tract
of
birds
with
respiratory
signs.
One
isolate
was
cultured
from
the
liver
of
a
dead
sharp
-tailed
munia
submitted
to
our
laboratory.
Since
A.
faecalis
strains
CCM
1052 and
NCIB
8156
showed
identical
pro-
perties
in
all
tests
conducted
only
the
results
of
CCM
1052
are
given.
Strain
B.
parapertussis
NCTC
5952
was
only
examined
for
electrophoretic
protein
patterns
and
was
not
included
in
the
other
tests.
Physical
characteristics
The
physical
characteristics
of
the
bacteria
studied
were
colonial
morphology
and
the
presence
or
absence
of
flagella.
Strains
grown
aerobically
in
a
humid
atmosphere
on
blood
and
veal
infusion
agar
(VI
agar)
were
examined
after
24
hours
incubation
at
35
°
C.
Flagella
staining
was
done
as
described
by
Walc-Polcrzywnicici
(1973)
by
use
of
bac-
terial
cells
harvested
from
blood
agar
cultures
after
24
hours
incubation
at
room
temperature.
Growth
characteristics
Inoculated
blood
agar
and
VI
agar
plates
were
incubated
aerobically
and
anaerobically
(BBL
-Gas
Pak
110
System)
for
2
and
5
days.
Salmonella-shigella
agar
(BBL)
and
MacConkey's
agar
(Merck,
Darmstadt)
were
pre-
pared
according
to
the
manufacturers'
directions.
Growth
was
recorded
after
5
days
incubation.
All
inoculated
plates
were
incubated
at
35
°
C.
Biochemical
tests
Oxidase
activity
was
tested
from
18
to
24
hours
cultures
with
a
freshly
prepared
1%
solution
of
tetramethylparaphenylenediamine
dihydorchloride
(Kovdcs
reagent)
(Sigma)
and
with
the
less
sensitive
solution
of
0.2
ml
1%
a
-naphthol
in
95%
ethanol
and
0.3
ml
1%
dimethyl-1,
4-phenylenediammonium
dichloride
(Merck,
Darmstadt)
(Gaby
and
Hadley,
1957).
Two
to
three
drops
of
the
reagent
were
placed
on
a
piece
of
fi
lter
paper
in
a
Petri
dish
and
then
some
colonies
were
removed
with
a
cover
slip
and
smeared
across
the
surface
of
the
impregnated
paper.
The
reactions
were
read
within
30
seconds
for
the
Kovdcd
reagent
and
within
2
minutes
for
Gaby
and
Hadley's
reagent.
Avian
Bordetella-like
strains
Table
1.
Reference
strains
compared
with
the
strains
in
Table
2.
265
Strain
designation
and
Source
a
Name
as
received
Remarks
C C
M
1052
b
N
C
1
B
8I56
b
C C
M
267
NCTC415
Alcaligenes
faecalis
A.
faecalis
A.
faecalis
A.
faecalis
=ATCC
8750
b
=
AT
C C
8750
b
NCTC
452
b
NCTC
454
NCTC456
NCTC
458
Ames
(NADC)
Bordetella
bronchiseptica
B.
bronchiseptica
B.
bronchiseptica
B.
bronchiseptica
B.
bronchiseptica
NCTC
8582
A.
denitrificans
Leifson
and
Hugh
(1954)
Holotype
K
M
543
Achromobacter
xylosoxidans
Yabuuchi
et
al.
(1974)
Type
strain
=NCTC
10807
NCTC
5952
b
B.
parapertussis
a
Abbreviations:
NCTC,
National
Collection
of
Type
Cultures,
London,
England;
CCM,
Czechoslovak
Collection
of
Microorganisms,
Brno,
Czechoslovakia;
NADC,
National
Animal
Disease
Center,
Ames,
Iowa;
KM
543,
obtained
from
K.
Kersters,
Laboratory
of
Microbiology
and
Microbial
Genetics,
State
University,
Gent,
Belgium;
NCIB,
National
Collection
of
Industrial
Bacteria,
Aberdeen,
Scotland.
b
Indicates
the
type
strain
in
the
Approved
Lists
of
Bacterial
Names
(Skerman
et
al.,
1980).
Table
2.
Non
fermentable
gram
-negative
bacterial
strains
from
birds
studied.
No.
of
isolates
.
Species
of
origin
Geographical
origin
Supplied
by
e
12
turkey
F
R
G
Own
isolates
1
chicken
F
R
G
Own
isolate
1
goose
F
R
G
Own
isolate
1
duck
F
R
G
Own
isolate
1
sharp
-tailed
munia
F
R
G
Own
isolate
2
turkey
Spain
M.A.
Dolz
2
turkey
U
S
A
R.B.
Rimler
1
turkey
U
S
A
D.G.
Simmons
4
turkey
Israel
U.
Bendheim
5
turkey
England
G.P.
Wilding
a
Addresses
M.A.
Dolz,
Division
Avese
ectas,
Tortosa,
Spain;
U.
Bendheim,
Institute
of
Poultry
Diseases,
Jerusalem,
Israel;
R.B.
Rimler,
National
Animal
Disease
Center,
Ames,
Iowa,
USA;
D.G.
Simmons,
School
of
Veterinary
Medicine,
North
Carolina
State
University,
Raleigh,
North
Carolina,
USA;
G.P.
Wilding,
Veterinary
Laboratory,
Chester,
England.
266
K.-11.
Hinz
et
al.
Catalase
activity
was
determined
using
VI
agar
cultures.
In
testing
for
catalase
a
few
colonies
were
removed
with
a
cover
slip
and
mixed
in
a
drop
of
3%
H202.
The
evolu-
tion
of
gas
bubbles
within
2
minutes
was
recorded
as
a
positive
test
for
catalase.
Most
of
the
other
biochemical
tests
were
performed
according
to
standard
methods
(Cowan,
1974).
Unless
otherwise
indicated
inoculated
test
media
were
incubated
at
35
°
C
in
aerobic
environment
for
5
days
and
were
read
daily.
Urea
hydrolysis
was
ob-
served
on
Christensen
urea
agar
(Merck).
Nitrate
reduction
was
determined
after
1,
2,
3,
4
and
5
days
of
incubation
in
nitrate
broth
(Merck)
and
the
isolates
were
tested
for
nitrate
reduction
with
Griess-Ilosvay's
reagent.
Negative
reactions
were
confirmed
by
addition
of
zinc
dust.
The
ability
to
utilise
citrate
as
a
carbon
source
was
tested
on
Simmon
citrate
agar
(Oxoid)
and
Christensen
citrate
agar
(Merck).
For
the
methyl
red
test,
the
MR
-VP
broth
(Merck)
was
used
and
cultures
were
tested
after
1
and
5
days
incubation
by
adding
methyl
red.
Indole
production
was
tested
in
Standard
H
broth
(Merck)
after
2
and
5
days
by
adding
1
ml
of
Kovici
indole
reagent
(Merck)
to
5
ml
broth
culture.
Gelatine
hydrolysis
was
tested
on
gelatin
agar
as
described
by
Cowan
(1974)
by
fl
ooding
the
plates
with
the
reagent
of
Frazier
(1926).
Aesculin
hydrolysis
was
determined
in
aesculin
broth
(Cowan,
1974).
Alkalinisation
of
litmus
milk
was
tested
in
steamed
milk,
containing
7%
Kubel-Tiemann's
litmus
solution
(Merck).
To
examine
the
oxidative
-fermentative
utilisation
of
glucose,
the
oxidation
-fermentation
medium,
of
the
Center
for
Disease
Control
(CDC)
pH
73
(Pickett,
1980)
with
brom-
thymol
blue
as
indicator
was
used
(2.5
ml
of
a
0.4%
aqueous
bromthymol
blue
solution
to
100
ml
medium).
Duplicate
tubes
were
inoculated
by
stabbing
with
a
straight
wire.
To
one
of
the
tubes
a
layer
of
molten
soft
paraffin
to
a
depth
of
1
cm
was
added.
Other
enzymes
(Table
5)
were
investigated
using
the
APIZYM
system
(API
Labor
System
GmbH,
Wiesbaden).
Overnight
cultures
on
VI
agar
were
washed
twice
and
sus-
pended
in
sterile
distilled
water
to
give
turbidity
of
tube
No.5
on
the
MacFarland
scale.
After
4
hours
at
37
°
C,
test
reagents
made
up
according
to
the
manufacturers'
instructions
was
added.
To
test
the
ability
of
the
strains
to
alkalinise
amides
and
or-
ganic
salts
(Table
6)
Greenwood's
low
peptone
(012)
medium
pH
6.5,
with
bromthy-
mol
blue
instead
of
phenol
red
as
an
indicator,
was
used
(Pickett,
1980).
Antibiotic
sensitivity
test
The
disc
method
of
the
antibacterial
sensitivity
test
was
conducted
on
D.S.T.
agar
(Oxoid).
Plates
were
incubated
at
35
°
C
for
16
to
18
hours
and
examined
for
zone
in-
hibition.
Haemagglutination
One
drop
of
a
heavy
bacterial
suspension
in
phosphate
-buffered
saline
(packed
bac-
terial
cell
volume
10%)
was
mixed
with
one
drop
of
20%
washed
chicken
erythrocytes
on
a
white
plastic
plate.
This
mixture
was
rotated
and
fi
nal
reactions
were
recorded
after
5
minutes.
Polyacrylamide
gel
electrophoresis
Soluble
proteins
obtained
by
sonication
of
bacterial
suspensions
were
compared
in
the
polyacrylamide
fl
at
gel
electrophoresis
using
sodium
dodecyl
sulphate
containing
gradient
gel
with
a
final
concentration
of
7.5
to
15%
acrylamide
(Laemmli,
1970).
The
apparatus
used
was
that
of
Studier
(1973)
modified
by
Neumann
and
Hinz
(1977).
About
20
pg
of
bacterial
protein/sample
gave
optimal
resolution.
Electrophoresis
was
run
at
room
temperature
at
a
constant
current
of
18
m
A;
the
voltage
was
50
volts
at
the
beginning
and
130
volts
at
the
end
of
the
run,
which
lasted
about
5
hours
(Romer,
Avian
Bordetella-like
strains
267
1982).
Gel
slabs
were
stained
with.Coomassie
blue
R
250
(Serva,
Heidelberg).
The
degree
of
interrelations
between
the
patterns
of
the
different
strains
are
expressed
numerically
as
percentage
congruence.
Percentage
congruence
=
2NAB
x
100
NA
+
NB
NA
and
NB
are
the
total
numbers
of
bands
visible
for
strains
A
and
B
and
NAB
is
the
number
common
to
both.
Pathogenicity
tests
These
experiments
were
done
to
determine
whether
or
not
the
avian
nonfermentable
strains,
B.
bronchiseptica
and
A.
faecalis,
are
pathogenic
for
turkey
poults.
All
the
strains
listed
in
Table
2,
two
B.
bronchiseptica
and
two
A.
faecalis
strains
were
tested.
For
these
tests,
1
-day
-old
turkey
poults
were
obtained
from
a
commercial
parent
breeder
flock
free
of
Mycoplasma
gallisepticum
and
Mycoplasma
synoviae.
No
agglu-
tinating
antibodies
against
the
thermolabile
antigen
of
the
bacterial
turkey
coryza
agent
could
be
detected
in
the
poults
from
this
fl
ock.
At
the
3rd
day
of
age
five
poults
were
inoculated
intranasally
with
about
10
4
colony
forming
units
(c.f.u.)
of
one
of
the
strains.
Immediately
after
inoculation
the
five
birds
were
placed
in
direct
-contact
with
5
to
10
uninoculated
poults.
Each
of
the
groups
was
kept
in
a
separate
negative
-
pressure
isolation
unit
80
x
150
x
80
cm
in
size
under
optimal
conditions.
The
poults
were
examined
for
clinical
signs
over
a
period
of
10
to
12
days.
The
examinations
in-
cluded
observation
and
gentle
manual
compression
of
the
upper
beak
in
the
region
of
nasal
turbinates.
After
killing,
the
poults
were
examined
for
macroscopic
lesions
and
the
tracheas
were
cultured
for
bacteria
by
scraping
the
mucosa
of
the
trachea
with
an
inoculating
loop
and
making
streaks
on
blood
agar.
RESULTS
All
the
bacterial
strains
from
turkeys
with
typical
properties
of
the
bacterial
turkey
coryza
agent
are
designated
as
Bordetella-like
agent
(BL
strains)
in
the
following
re-
sults.
Colonial
morphology
and
motility
As
reported
earlier
(Hinz
et
al.,
1978)
the
BL
bacterium
is
a
capsulated
gram
-negative
rod
with
an
average
size
of
0.4
to
0.5
pm
by
1
to
2
pm.
When
grown
on
blood
agar
and
VI
agar
two
distinct
types
of
colony
were
recognised
among
the
BL
strains.
The
most
notable
differential
characteristics
were
colony
size
and
configuration.
One
type
was
small,
compact
and
pearl
-like,
with
an
entire
edge
and
glistening
surface,
measuring
<
1
mm
in
diameter
after
24
hours
of
incubation
(type
I).
The
other
type
was
larger,
circular,
convex
with
an
entire
edge
and
smooth
surface
(type
II).
The
type
I
colonies
often
had
a
brownish
centre
on
VI
agar.
When
grown
on
VI
agar
a
third
type
(not
included
in
this
study)
differed
from
type
II
by
its
serrated
irregular
edge
and
greater
colony
size.
Type
I
can
dissociate
to
type
II
and
type
II
to
III.
No
reversion
has
been
observed
in
vitro
and
in
vivo.
Only
three
BL
strains
showed
dissociation.
The
other
25
remained
stable
in
the
given
morphological
state
for
several
passages.
The
colonies
of
strain
P
4083
differed
from
the
BL
strains
in
that
they
were
larger
with
a
raised
centre,
spreading
perimeter,
and
irregular
edge
and
surface.
The
A.
faecalis
reference
strains
used
showed
the
same
type
of
colony
morphology
as
P
4083.
Strain
40
-
81
produced
colonies
of
the
intermediate
type
on
VI
agar
as
des-
cribed
by
Bemis
et
al.
(1977).
268
K.
-H.
Hinz
et
al.
All
BL
strains
were
motile.
On
average,
5
to
8
peritrichously.arranged
fl
agella
per
rod
could
be
observed.
The
same
type
of
flagellation
was
found
in
the
reference
strains
(Table
1).
The
number
of
motile
rods
was
significantly
greater
in
cultures
of
the
BL
strains
incubated
at
room
temperature
than
in
those
cultured
at
35
°
C.
This
phenome-
non
was
not
found
for
the
reference
strains.
Cultural
properties
The
results
of
the
tests
are
presented
in
Table
3.
The
A.
faecalis
reference
strains
and
P
4083
grew
as
swarming
colonies
on
VI
agar
under
anaerobic
condition.
All
strains
tested
were
able
to
grow
not
only
at
35
°
C
but
also
at
42
°
C,
although
some
reference
strains
grew
poorly
at
42
°
C.
Table
3.
Growth
characteristics
and
other
biochemical
reactions
of
the
avian
Bordetella-like
bacterium
(BL)
and
two
isolates
from
turkeys
(P
4083
and
4081)
compared
with
those
of
the
reference
strains
of
B.
bronchiseptica
(BB),
A.
faecalis
(AF),
A.
denitrificans
(AD)
and
Achromobacter
xylosoxidans
(A
X).
Test
a
L
(n
B
=28)
B
(n
B
=
5)
AF
(n=3)
AD
NCTC
8582
AD
KM
543
40-81
P
4083
Aerobic
growth
+
(28)
+
(5)
+
(3)
+
+ +
+
Anaerobic
growth
(
0)
(0)
w
(3)
w
Growth
on
MacConkey's
agar
+
(28)
+
(5)
+
(3)
+
+ + +
Growth
on
Salmonella-shigella
agar
+
(28)
+
(5)
+
(3)
+
+
+ +
Kovdcs
oxidase
+
(28)
+
(5)
+
(3)
+
+
+ +
Gaby-Hadley's
oxidase
—/w(2)
+
(5)
+
(3)
+ +
+ +
Catalase
+
(28)
+
(5)
+
(3)
+
+ + +
Urease
(
0)
+
(5)
(0)
+
Nitrate
reduction
(
0)
+
(5)
(0)
+
+ +
Citrate
as
C
source
+
(28)
+
(5)
+
(
3
)
+
+ +
+
Methyl
red
(
0)
(0)
(0)
Indole
-
(
0)
(0)
(0)
Gelatin
liquefaction
(
0)
(0)
(0)
Aesculin
hydrolysis
(
0)
(0)
(0)
Alkalinisation
of
litmus
milk
(
0)
+
(5)
+
(3)
+
+ +
+
0/F
(Glucose):
Fermentative:
Acid
(
0)
(0)
(0)
Alkali
(
0)
(0)
(0)
Oxidative:
Acid
(
0)
(0)
(0)
+
Alkali
(
0)
(0)
(0)
Acid
from
carbohydrates*
(
0)
(0)
(0)
+
Xylose
Sensitivity
to
Penicillin
G
2
Units/disc
—/+(9)
(0)
(0)
10
Units/disc
+/—(27)
(0)
(0)
Haemagglutination
of
chicken
erythrocytes
(
0)
—/+(1)
(0)
+
a
Symbols:
+
=
positive
result;
=
negative
result;
w
=
weak
or/and
delayed
positive;
Number
in
parentheses
are
number
of
strains
positive.
*No
acid
was
produced
from
maltose,
arabinose,
inositol,
saccharose,
mannitol
and
lactose.
Avian
Bordetella-like
strains
269
Biochemical
characteristics
The
test
results
are
summarised
in
Tables
3,4
and
5.
All
the
reference
strains
(Table
1),
strains
40
-
81
and
P
4083,
gave
positive
reactions
for
oxidase
with
the
Kovdcd
re-
agent
within
10
seconds,
whereas
the
BL
strains
reacted
only
after
10
to
30
seconds.
The
BL
strains
clearly
differed
from
the
other
strains
when
Gaby
and
Hadley's
test
reagent
was
used.
Only'two
BL
strains
from
Israel
gave
weak
delayed
reactions
with
the
latter
reagent.
However,
more
of
the
BL
strains
gave
positive
oxidase
reactions
when
48
and
72
hour
cultures
were
used.
BL
strains
could
also
be
distinguished
by
their
inability
to
alkalinise
litmus
milk
and
by
their
sensitivity
to
10
unit
penicillin
discs.
The
properties
of
strains
40
-
81
and
P
4083
were
similar
to
those
of
B.
bron-
chiseptica
and
A.
faecalis
respectively.
Relevant
differences
in
respect
to
enzyme
activity
in
the
API-ZYM-test
system
were
not
detected
(Table
4).
The
production
of
alkali
from
amides
and
organic
salts
are
given
in
Table
5.
Reactions
of
strains
40
-
81
and
P
4083
were
also
similar
to
those
of
B.
bronchiseptica
and
A.
faecalis
respec-
tively.
The
BL
strains
attacked
fewer
substrates
than
those
strains
listed
in
Table
1.
They
were
different
from
B.
bronchiseptica,
A.
faecalis,
A.
denitrificans,
Achromo-
bacter
(Achr.)
xylosoxidans
and
the
strains
40
-
81
and
P
4083
by
their
lack
of
alkalinisation
of
malonamide,
valeramide
maleate,
malonate
and
valerate.
Electrophoretic
protein
patterns
The
electrophoresis
of
soluble
proteins
show
that
our
own
BL
strains
isolated
from
various
species
of
birds,
and
the
BL
strains
obtained
from
USA,
Israel,
Spain
and
England,
display
very
similar
and
almost
identical
protein
patterns.
The
protein
patterns
of
four
representative
avian
BL
strains
are
shown
in
Fig.1
(48
to
50
bands
per
strain).
The
protein
pattern
of
BL
strain
591
-
77
clearly
differs
from
those
of
B.
bronchiseptica,
B.
parapertussis,
A.
faecalis,
A.
denitrificans,
Achr.
xylosoxidans
and
of
strains
40
-
81
and
P
4083
(Fig.2).
In
Table
6
the
degree
of
relationship
be-
tween
the
avian
BL
group
and
the
other
bacteria
tested
is
presented
numerically
as
percentage
congruence.
Strains
40
-
81
and
P
4083
exhibited
the
highest
degree
of
Table
4.
Enzyme
activities
of
B.
bronchiseptica
(BB),
A.
faecalis
(AF),
A.
denitrificans
(AD),
Achromobacter
xylosoxidans
(Ax)
and
selected
avian
Bordetella-like
strains
(BL)
tested
in
the
A
PI-ZYM
system.
Enzymes
a
BL
n=8
BB
n=5
AF
n=3
AD
n=1
AX
n=1
Alkaline
phosphatase
w
(8)
w
(5)
w
(3)
- -
Esterase
+
(8)
+
(5)
+
(3)
+ +
Esterase
li
pase
w
(8)
w
(5)
w
(3)
- -
Lipase
-
(0)
-
(0)
-
(0)
-
-
Leucine
arylamidase
+
(8)
+
(5)
+
(3)
+ +
Valine
arylamidase
-
(0)
-
(0)
-
(0)
- -
Cysteine
arylamidase
-
(0)
-
(0)
-
(0)
-
-
Trypsin
-
(0)
-
(0)
-/w
(1)
-
_
Chymotrypsin
-
/w
(5)
-
(0)
-
/w
(1)
-
-
Acid
phosphatase
+
(8)
+
(5)
+
(3)
w
+
Phosphoamidase
w
(8)
w
(5)
w
(3)
-
w
a
Symbols
used:
+
=
strains,
positive;
=
negative;
w
=
weak
positive;
(
)
number
of
strains
positive;
All
the
strains
tested
were
negative
for
a-galactosidase,
P-galactosidase,
a-glucuronidase,
a-glucosidase,
P-glucosidase,
N-acetyl-(jIlucosamidase,
a-mannosidase
and
a-fucosidase.
270
K.
-H.
Hinz
et
al.
Table
5.
Alkalinisation
of
amides
and
organic
salts
by
the
avian
Bordetella-
like
bacterium
(BL),
two
isolates
from
turkeys
(P
4083
and
40-81)
and
the
reference
strains
of
B.
bronchiseptica
(BB),
A.
faecalis
(AF),
A.
denifrificans
(AD)
and
Achromobacter
xylosoxidans
(AX).
Substrates'
BL
(n=28)
BB
(n=
5
)
AF
NCTC
(n=3)
AD
8582
AX
KM
543
40-81
P
4083
Acetamide
+
(28)
(0)
+
(3)
+ +
+
Asparagine
+
(28)
+
(5)
+
(3)
+
+ +
+
n-Butyramide
—/w(14)
+
(5)
+
(3)
+
+
+
+
Formamide
+
(28)
(0)
+
(3)
+ +
+
Glutamine
+
(28)
+
(5)
+
(3)
+ + +
+
Malonamide
(
0)
+
(5)
+
(3)
+
+
+ +
Propionamide
+
(28)
+
(5)
+
(3)
+
+
+ +
Succinamide
+
(28)
+
(5)
+
(3)
+
+
+ +
Valeramide
(
0)
+/w(5)
+/w(3)
+
+
+ +
Acetate
+
(28)
+
(5)
+
(3)
+ +
+
+
Adipate
+
(28)
+
(5)
(0)
+ + +
+
Citrate
+
(28)
+
(5)
+
(3)
+
+ + +
Maleate
(
0)
+
(5)
+
(3)
+
+
+
+
Malonate
(
0)
+
(5)
+
(3)
+
+
+
+
Mucate
(
0)
+/—(3)
(0)
+
+ +
Propionate
+
(28)
+
(5)
+
(3)
+
+
+
+
Saccharate
(
0)
+/—(4)
(3)
+ +
+
Valerate
(
0)
w/+(5)
w/+(3)
+
+
+
Formate
+
(28)
+
(5)
+
,(3)
+
+
+
+
a
Symbols:
+ =
positive
result;
=
negative
result;
w
=
weak
or/and
delayed
positive;
Number
in
parentheses
are
number
of
strains
positive.
similarity
to
B.
bronchiseptica
and
A.
faecalis
respectively.
However,
the
similarity
of
strain
40
-
81
to
the
NCTC
B.
bronchiseptica
strains
was
far
less
than
to
B.
bronchi-
septica
strain
Ames.
The
similarity
of
the
BL
strains
to
B.
bronchiseptica
and
strain
40
-
81
was
marked
higher
than
to
A.
faecalis
or
to
the
other
bacteria
tested.
Pathogenicity
in
turkey
poults
All
the
28
avian
BL
strains
and
each
of
two
strains
of
B.
bronchiseptica
and
A.
faecalis,
as
well
as
the
isolates
40
-
81
and
P
4083,
were
tested.
All
the
BL
strains
were
able
to
produce
a
respiratory
disease
with
the
clinical
picture
of
turkey
coryza.
The
main
signs
consisted
of
accumulation
of
mucus
on
the
external
nares,
a
foamy
conjunctivitis
and
oedema
of
the
paraorbital
region.
However,
the
clini-
cal
signs
varied
substantially
in
severity.
In
mild
disease
the
only
clinical
signs
initiated
was
excess
mucus
production
in
the
nasal
cavity
and
this
could
often
be
seen
only
when
pressure
was
applied
to
upper
beak
in
the
region
of
nasal
turbinates.
The
BL
organisms
could
be
reisolated
from
the
tracheas
of
all
the
inoculated
and
nearly
all
the
direct
-contact
exposed
birds.
Table
6.
Degree
of
relationship
between
the
cell
protein
electrophoretic
patterns
of
different
strains
expressed
numerically
as
percentage
congruence.
Strains
BL
(n
=
28)
BB
(n
=
4)
AF
(n
=
3)
AD
NCTC
8582
AX
KM
543
40-81
BB
Ames
P
4083
BP
NCTC
5952
Bordetella-like
(BL)
94-100
a
B.
bronchiseptica
NCTC
(BB)
63—
69
90-90
A.
faecalis
CCM
and
NCTC
(AF)
40—
47
42-58
73-79
a
A.
denitrificans
NCTC
8582
51—
53
55-61
50-52
100
Achr.
xylosoxidans
KM
543
50—
51
51-57
49-57
73
100
Turkey
isolate
40-81
70—
72
79-85
50-58
58
56
100
B.
bronchiseptica
Ames
63—
65
76-87
48-55
60
51
93
100
Turkey
isolate
P4083
44—
48
47-52
75-83
51
51
51
54
100
B.
parapertussis
NCTC
5952
(BP)
60—
61
67-71
41-52
58
52
66
63
44
100
a
Percentage
congruence
of
patterns
between
the
same
strains
was always
100.
Avian
Bordetella-like
strains
272
K.-11.
Hinz
et
a/.
DISCUSSION
With
the
exception
of
two
strains,
the
other
28
strains
listed
in
Table
2
form
a
tight
taxon
with
characteristics
of
the
avian
gram
-negative
nonfermentable
bacterium
tenta-
tively
designated
as
a
Bordetella
like
agent
by
Hinz
et
al.
(1978).
The
remaining
two
strains
40
-
81
and
P
4083
had
the
typical
characteristics
of
B.
bronchiseptica
and
A.
faecalis
respectively.
Simmons'
classification
of
the
bacterial
agent
as
A.
faecalis
was
not
confirmed
in
this
study
because
one
of
the
strains
submitted
by
him
as
repre-
sentative
of
the
bacterial
agent
of
turkey
coryza
possesses
properties
typical
of
those
of
the
BL
bacterium.
On
the
basis
of
the
properties
found,
none
of
the
BL
strains
could
be
assigned
taxonomically
to
B.
bronchiseptica,
A.
faecalis,
A.
denitrificans,
Achr.
xylosoxidans
or
B.
parapertussis.
Since
the
cell
proteins
are
genetically
directed,
their
patterns
obtained
by
polyacrylamide
gel
electrophoresis
tend
to
express
genetic
relationship
between
microorganisms
(Norris,1980).
For
that
reason
the
differences
of
protein
patterns
between
the
nonfermentable
strains
tested
make
it
very
probable
that
the
BL
strains
are
a
separate
species.
The
mol
%
G
+
C
values
of
DNA
and
the
results
of
extended
ribosomal
RNA.
DNA
hybridisations
support
a
separate
taxono-
mix
position
in
the
genus
Bordetella
(Kersters,
Segers
and
Hinz,
to
be
published).
The
isolation
of
BL
strains
not
only
in
Germany
and
USA,
but
also
in
Spain,
Israel
and
England,
indicate
that
this
bacterial
agent
is
possibly
worldwide
in
distribution.
Hitherto
the
BL
agents
have
been
isolated
mainly
from
turkeys.
However,
with
iso-
lates
from
turkeys
it
was
possible
to
produce
a
turkey
coryza-like
disease
in
ducks
(Hinz
et
al.,
1979b)
and
young
Japanese
quail
(Simmons
and
Gray,
1979).
The
isola-
tion
of
the
BL
bacterium
from
a
chicken
and
from
ducks
was
reported
by
Hinz
et
al.
(1979a).
Recently
Simmons
et
al.
(1981)
have
isolated
the
BL
agent
from
eight
of
10
broiler
chick
fl
ocks
with
signs
of
a
respiratory
disease.
The
clinical
picture
of
turkey
coryza
could
be
produced
in
healthy
chicks
and
turkey
poults
with
chick
isolates.
The
ability
of
the
BL
strains
isolated
from
a
chicken,
goose,
duck
and
sharp
-tailed
munia
to
produce
a
respiratory
disease
with
the
clinical
picture
of
turkey
coryza
in
turkey
poults
under
experimental
conditions
can
be
interpreted
as
an
indication
of
the
wide
host
spectrum
of
the
BL
agent
within
the
class
of
Ayes.
On
the
basis
of
the
results
presented,
identification
of
the
BL
bacterium
is
possible
without
difficulties.
Differences
in
the
biochemical
features,
pathogenicity
and
electrophoretic
protein
patterns
have
not
been
observed
between
colony
types
I
and
II
of
the
BL
strains.
However,
Hertle
(1981),
who
has
examined
serologically
some
of
the
BL
strains,
found
relationships
between
surface
antigenic
structure and
the
type
of
colony
on
agar
plates.
Acknowledgement
The
authors
thank
Rita
Leise
for
her
excellent
technical
assistance.
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RESUME
Etude
comparee
de
souches
pseudo-Bordetella
aviaires,
de
Bordetella
bronchiseptica,
d'Alcalig4nes
faecalis
et
d'autres
bacteries
apparentees
non
fermentantes
Les
caracteristiques
culturales
et
bio-chimiques,
les
modeles
electrophoretiques
et
le
pouvoir
pathogene
chez
la
dinde
de
30
souches
de
bacteries
aviaires
gram
-negatives
non
fermentantes,
obtenues
dans
differentes
regions
geographiques,
parmi
lesquelles
26
ont
ete
isolees chez
la
dinde
et
les
autres
a
partir
du
poulet,
du
canard,
de
l'oie
et
du
domino
d
longue
queue
ont
ete
comparees
d
Bordetella
bronchiseptica,
Alcaligenes
faecalis,
Alcaligenes
denitrificans,
Achromobacter
xylosoxidans
et
Bordetella
para-
pertussis.
A
l'exception
de
deux
souches
isolees
chez
la
dinde,
toutes
les
souches
non
276
K.
-H.
Hinz
et
al.
fermentantes
ont
constitue
un
groupe
tres
homogene
ayant
les
caracteristiques
de
1'agent
du
coryza
de
la
dinde
qui
avait
ete
primitivement
designe
comme
une
pseudo-
Bordetella.
Sur
la
base
des
criteres
etudies
elle
pourrait
etre
clairement
differenciee
de
B.
bronchiseptica,
A.
faecalis
et
des
autres
bacteries,
suggerant
que
la
pseudo-
Bordetella
occupe
une
position
taxonomique
distincte.
Aucune
difference
n'a
ete
trouvee
entre
les
souches
de
coryza
de
la
dinde
isolees
en
Allemagne
et
celles
isolees
aux
Etats-Unis.
Les
deux
souches
de
bacteries
restantes,
isolees
chez
la
dinde,
ont
montre
les
caracteristiques
typiques
de
Bordetella
bronchiseptica
et
A.
faecalis
res-
pectivement.
ZUSAMMENFASSUNG
Vergleichende
Untersuchungen
von
aviaren
Bordetella-iihnlichen
St
ammen,
Bordetella
bronchiseptica,
Alcaligenes
faecalis
und
anderen
verwandten
nichtfermentativen
Bakterien
Die
kulturellen
und
biochemischen
Eigenschaften,
die
Elektrophoreseprofile
loslicher
Proteine
sowie
die
Pathogenitit
fiir
Putenkiiken
von
30
gramnegativen
und
nichtfer-
mentativen
Bakterien-Sammen
unterschiedlicher
geographischer
Herkunft,
von
denen
26
aus
Puten
und
je
eines
aus
dem
Huhn,
der
Ente,
der
Gans
und
einem
japan-
ischen
Mowchen
isoliert
worden
waren,
wurden
mit
jenen
von
Bordetella
bronchi-
septica,
A.
faecalis,
Alcaligenes
denitrificans,
Achromobacter
xylosoxidans
und
Bordetella
parapertussis
verglichen.
Mit
Ausnahme
von
zwei
Putenisolaten
bildeten
die
anderen
aviiren
nichtfermenta-
tiven
Stimme
ein
sehr
homogenes
Taxon
mit
Eigenschaften
des
bakteriellen
Puten-
schnupfenerregers,
der
voraufig
als
ein
Bordetella-ahnliches
Bakterium
bezeichnet
worden
ist.
Auf
der
Basis
der
ermittelten
Charakteristica
konnten
die
Bordetella-
ahnlichen
Bakterien
eindeutig
von
B.
bronchiseptica,
A.
faecalis
und
den
anderen
getesteten
Bakterien
unterschieden
werden,
was
daffir
spricht,
daf3
das
Bordetella-
ahnliche
Bakterium
eine
separate
taxonomische
Position
einnimmt.
Keine
relevanten
Unterschiede
wurden
zwischen
den
in
Deutschland
isolierten
Stimmen
und
jenen
aus
den
USA
gefunden.
Die
ilbrigen
beiden
Bakterien-Stimme
aus
Puten
zeigten
die
typischen
Eigenschaften
der
Species
B.
bronchiseptica
bzw.
A.
faecalis.