Diagnosis of Avian Tuberculosis-mycobacteriosis by Rapid Agglutination

Abstract

In contrast to the intradermal tuberculin test (ITT) the sensitivity and specificity of rapid agglutination (RA) using the M. avium serovar 2, 3 antigen increased at advanced stages of the infection. Only 47.7% of pullets, infected orally with 1 mg of a culture of a virulent strain of M. avium serovar 2 per 1 kg body mass, reacted to avian tuberculin, while RA yielded, at the sama stage of infection, 61.6% and 90.9% of positive results when whole blood and blood serum were examined, respectively. Birds reacting to avian tuberculin were found in some avian tuberculosis-free farms. Contamination of sawdust, used as the litter, with M. intracellulare serovar 8 was a frequent cause of such reactions. RA with M. avium antigen (MAA) was also used successfully for the differentiation of paraallergic from specific reactions to avian tuberculin. Parallel examination by ITT and RA is recommended as a component of programmes of avian tuberculosis elimination. Moreover RA can replace ITT in screening of tuberculosis in poultry flocks and bird colonies. Examination of blood sera, yielding more accurate results, in recommended in birds kept in zoological gardens and in rearing colonies of predatory birds. RA with MAA is more specific than ITT. Considering the existence of pathogenic strains of M. avium which cannot be classed with any of the reference serovars 1, 2 and 3, continuous attention should be paid to the selection of avian mycobacterial strains for the production of the antigen.

Full text

•
ACTA
VET.
BRNO,
62, 1993:
63-69-
DIAGNOSIS OF AVIAN TUBERCULOSIS-MYCOBACTERIOSIS
BY
RAPID AGGLUTINATION
M.
PAVLAS,*
Alena
MICHALSKA,
M.
HUNADY
*Veterinary
Research
Institute,
621
32
Brno
Bioveta,
68323
Ivanovice
na
Hane
Received September 7, 1992
Abstract
Pa
vIas
M.,
Alena
Michalska,
M.
H
unady:
Diagnosis
of
Avian
Tubercu/osis-
• -Mycobacteriosis
by
Rapid Agglutination.
Acta
vet.
Brno,
62, 1993:
63-69.
In
contrast
to
the
intradermal
tuberculin
test
(ITT)
the
sensitivity
and
specificity
of
rapid
agglutination eRA)
using
the
M.
avium serovar 2, 3
antigen
increased
at
advanced stages
of
the
infection.
Only
47.7%
of
pullets, infected orally
with
1
mg
of
a
culture
of
a
virulent
strain
of
M.
avium serovar 2
per
1
kg
body
mass,
reacted
to,
avian
tuberculin,
while
RA
yielded,
at
the
sama stage
of
infection, 61.6%
and
90.9%
of
positive results
when
whole
blood
and
blood
serum
were examined, respectively.
Birds reacting
to
avian
tuberculin
were
found
in
some avian tuberculosis-free farms.
Contamination
of
sawdust,
used
as
the
litter,
with
M.
intracellulare serovar 8 was
a
frequent
cause
of
such
reactions.
RA
with
M.
avium antigen
(MAA)
was also
used
successfully
for
the
differentiation
of
paraa1lergic
from
specific reactions
to
avian
tuberculin.
Parallel examination
by
ITT
and
RA
is
recommended
as a
component
of
programmes
of
avian tuberculosis elimination.
Moreover
RA
can
replace
ITT
in
screening
of
tuberculosis
in
poultry
flocks
and
bird
colonies. Examination
of
blood
sera, yielding
more
accurate results,
in
recommended
in
birds
kept
in
zoological
gardens
and
in
rearing
colonies
of
predatory
birds.
RA
with
MAA
is
more
specific
than
ITT.
Considering
the
existence
of
pathogenic strains
of
M.
avium
which
cannot
be
classed
with
any
of
the
reference serovars
1,
2
and
3,
continuous
attention
should
be
paid
to
the
selection
of
avian mycobacterial strains for
the
production
of
the
anti-
gen.
Poultry, free-living birds, captive birds, diagnosis, avian tuberculosis, mycobacteriosis,
rapid agglutination
•
After
the
eradication
of
bovine tuberculosis
from
Czechoslovakia
in
1968, Mycobacterium-
avium has become
the
most
important
pathogenic mycobacterium infecting
not
only farms animals,..
but
also free-living
birds
and
mammals.
It
is
well established
that,
as
far
as domesticated bird&
are
concerned, avian tuberculosis affects
more
frequently chickens
than
pigeons
or
water
fowl.
Occurrence
of
tuberculosis
in
gallinaceous
birds
is
usually associated
with
extensive
poultry-
husbandry.
The
relationship
between
poultry
husbandry
methods
and
infection
rate
was
reported
by
many
authors.
The
current
replacement
of
extensive
husbandry
by
the
all-in-all-out systems
not
only
in
large
poultry
operations,
but
also
in
smaller farms, is accompanied
by
a
retreat
of'
avian tuberculosis. .
Despite
considerable variations
of
reliability,
the
intradermal
tuberculin
test
(ITT)
remains.
the
most
frequently
used
method
of
diagnosis
of
avian tuberculosis-mycobacteriosis.
The
reliability'
of
detection
of
birds
infected
with
M.
avium decreases especially
in
flocks
with
high
infection
rates,
and
as
postulated
by
numerous
authors,
the
sensitivity
of
ITT
may
be
as
low
as
50
to
60%.
Consequently, culling
of
reactors does
not
always
result
in
a complete elimination
of
the
infection,
although general epidemiological rules have
been
observed.
The
low reliability
of
ITT
motivated
the
search for
methods
which
would replace
it,
or,
at
least, give
more
precision
to
its results. One-
of
the
candidates is
rapid
agglutination (RA)
with
M.
avium
antigen
(MAA).
Gallinaceous
birds
have
been
regarded
as
the
major
primary
source
of
Mycobacterium avium-
•
for
a long period.
Numerous
investigations,
performed
in
Czechoslovakia
and
abroad,
demons--

'64
-trated avian tuberculosis also
in
free-living
bird
species, such as sparrows, bulls, ducks, jackdaws, •
pheasants, rooks, kestrels, owls,
and
eagles
(HejUl!ek
and
Balat
1973). Avian tuberculosis
.is often diagnosed
in
exotic birds kept
in
zoological gardens.
Both
in
vivo
and
post mortem methods are used for
the
diagnosis
of
avian tuberculosis. X-ray
-and haematological examinations are the clinical methods usable
on
captive birds
(Klime§
and
Pavlas
1961).
More
reliable results can be obtained
by
endoscopy, completed by bacterioscopic, histological
-and
or
bacteriological examination
of
bioptic samplex. Moreover, faeces
and
blood samples can
be
examined
by
culture.
The
probability
of
detection
of
mycobacteria is higher
in
birds than
in
mammals
(Hartwig
1965;
Roznyatovska
1975).
ITT,
introduced
in
1913, does
not
meet fully
the
requirements for sensitivity
and
specificity.
Low
reliability
of
ITT
was reported
by
numerous authors
(Fritzsche
and
Unruh
1958;
Prochorov
1955).
The
failure
to
detect a
M.
mnum
infection aften results from anergy accompa-
nying
generalized tuberculosis.
On
the
other
hand,
non-specific reactions
to
avian tuberculin
are frequent
in
poultry
and
other animal species
(Rozanska
1971).
The
first description
of
RA
~for
the
demonstration
of
tuberculosis
in
poultry was published
by
Moses
at
al. (1943) who
examined blood serum samples.
Karlson
et
a1.
(1950) developed the agglutination test for
the
·examination
of
whole blood samples.
Numerous reports
on
the
use
of
RA
for
the
diagnosis
of
tuberculosis
in
poultry
and
other
bird
:species were published
in
the
second
half
of
this century
(prochorov
and
Akulov
1958;
Ber-
.ger
1962;
Svrl!ek
1962;
Nassal
1963;
Betke
et
al. 1964;
Richter
1965;
Hiller
et
al. 1967; •
G6tz
1984;
Neumann
1988).
The
test is based
on
the
reaction
of
humoral antibodies with
,·corpuscular antigens, represented
by
a suspension
of
mycobacteria.
Under
optimci conditions,
the
agglutination takes place within several seconds.
The
simple test has become very popular
:and estimates
of
reliability
of
examinations
of
whole blood samples range from
80%
for spontaneo-
-usly infected
to
95%
for experimentally infected birds, respectively
(Havlik
1960;
Pavlas
1962).
High
specificity
and
sensitivity
of
RA
were also reported
by
G6tz
(1984), who found tuberculo-
:sis
in
only 2 (0.6%)
of
328 necropsied zoo birds reacting negatively
in
RA.
Materials
and
Methods
The
strain 583
of
M.
mnum
serovar
2,
3 was selected for
the
production
of
the
antigen from
:a
set
of
field strains isolated from
hen
affected with
the
nodular form
of
tuberculosis.
The
selection
--was
based
on
results
of
examinations
of
antigenic, biological, biochemical
and
growth character-
.istics.
The
serum-containing liquid medium for mycobacteria, supplied
by
the
Institute
of
Sera
and
Vaccines, Prague, proved
to
be
the best
of
the available solid
and
liquid media for
the
propagation
<of
M.
Q'lIium.
A well grown culture was centrifugated
and
washed twice with physiological saline
-with subsequent centrifugation.
One
part
of
the
final sediment was resuspended
in
25 parts
of
buffered physiological saline containing 0.5%
of
phenol.
The
suspension was left
to
stand
at
22°C
for 7 days with daily shaking.
The
density was measured spectrophotometrica1ly
at
605
mm
:and adjusted
to
15°C
to
20 °C.
The
antigen was checked for specificity
and
sterility,
and
sensitivity
activity checks were done using homologous
and
heterologous sera
of
rabbits immunized with •
lItrains
of
M.
mnum
and
M.
intracellulare, respectively.
RA was carried
out
using serial twofold dilutions
(1
: 2 - 1 : 64)
of
the
standard sera. Eight
",1
<of
the
antigen
and
2
",1
of
the
respective serum dilution were pipetted
on
to
a glass plate
and
the
"two
drops were combined carefully with a platinum loop.
The
plate was transluminated
and
the
results were read after 20
to
60 seconds.
The
antigen was considered active
if
a marked agglutin-
;ate was formed with
the
positive serum dilution 1 : 8 within 60 seconds.
No
agglutination was
observed with
any
of
the heterologous sera
or
with physiological saline.
The
sensitivity
and
specificity
of
the
M.
avium antigen were tested in poultry
and
pheasant
flocks free
of
or
infected with avian tuberculosis-mycobacteriosis as evidenced
by
complex examina-
'lions.
Results
As demonstrated
in
poultry flocks infected with tuberculosis, the diagnostic
:reliability
ofRA,
but
not that
ofITT,
increased
at
advanced stages
of
the
infection
<Fig. I).
•

•
•
65
1.00
90
80
,.
...
-
70
:>
...
...
...
60
-.
.c:
•
411
50
Coo
0
40
...
c:
•
30
0
So
•
At
20
1.0
0
A
B
C
Seo...
ot
the
1
••
Ions
The
reliability
of
both RA and
ITT
depended
not
only
on
the quality
of
the
antigen or allergen,
but
also on the way and stage
of
infection and on the virulence
of
the inoculated mycobacteria. Higher sensitivities
of
both tests were observed
in
experimentally infected birds.
ITT
and whole blood RA were positive
in
the
6
th
week after subcutaneous infection
in
85% and 80%
of
the birds, respectively.
On
the
other hand, 55,8%, 41,9% and 95,2%
of
pullets infected orally with
a virulent strain
of
M.
a'lJium
serovar 2 reacted positively at the same stage
of
the
infection in
ITT,
whole blood RA and blood serum RA, respectively.
The
highest
specificity
was
recorded in birds reacting positively both in
ITT
and in RA.
Results
of
ITT
and RA and post mortem finding in
108
hens from a
flock
infected
with avian tuberculosis are presented in Table
1.
• False positive reactions to avian tuberculin occur not only in cattle and swine,
but
were observed also in avian tuberculosis-free poultry
flock~.
The
paraallergic
reactions were often induced by M.
intracellulare,
contaminating sawdust to be
used for littering. Marked positive reactions to avian tuberculin were also observed
in
poultry infected experimentally with M.
intracellulare.
Positive reactions
in
ITT
were recorded
in
57,1
% and 97,1%
of
pullets infected intramuscularly with
this species. Simultaneous
ITT
with specific sensitins, prepared from the atypical
mycobacteria M.
intracellulare
and M.
a'lJium
failed to identify the type
of
allergy.
Of
the birds sensibilized with M.
intracellulare,
74,6% and 68,6% reacted to
specific sensitin and avian tuberculin, respectively.
The
corresponding values
for the test repeated after 6 weeks were 88,2% and 82,3%, respectively.
On
the
other hand, whole blood
RA, carried out parallelly with the repeated
ITT,
yielded
61,8% and 7,2%
of
positive results, respectively (Table 2).
The
results have shown that the two antigens can be used for the differentiation
of
allergies induced
by
any
of
the two mycobacterial species. This conclusion
• was also confirmed by investigations
in
poultry farms from which paraallergic .

66
reactions to avian tuberculin were reported. Fifty percent
of
3-month-old pullets •
reacted positively
in
ITT
to avian tuberculin
in
a large farm and twenty reactors
were examined by whole blood
RA.
While none
of
them reacted in the test with
the M.
avium
antigen,
15
were positive when the M.
intracellulare
antigen was
used (Table 3). M.
intracell~lare
serovar 8
was
isolated from sawdust to be used
for littering in the farm. Blood serum RA demonstrated the presence
of
agglutinins
more reliably
than whole blood RA
in
birds infected experimentally with
M.
intracellulare.
Positive reactions were obtained in 62,9% and 92,6%
of
the
birds
examined by whole blood RA and blood serum RA, respectively.
The
examina-
tion
of
blood sera reduced the number
of
non-specific reactions
in
RA
with
M.
avium
antigen to one
half
(Table 4).
RA failed to differentiate among birds infected experimentally with M.
intra-
cellulare
serovars 4, 3a,
6-9,
or 5.
Discussion
The
reliability
of
RA in the diagnosis
of
avian tuberculosis-mycobacteriosis> •
postulated by other authors, has been confirmed and completed by the finding
that RA can be used for the differentiation
of
allergic reactions to avian tuberculin
induced by non-virulent serovars
of
the M.
a'{)ium~intracellulare
complex.
It
has been demonstrated that the replacement
of
whole blood by blood serum.
increases the
sensitivity and specificity
of
RA.
The
two parameters depend
alS()
on the selection
of
a suitable strain for the production
of
the mycobacterial antigen
as
well
as
of
a culture medium supporting the formation
of
surface antigens>
which are decisive for the activity and specificity
of
the product.
Table
1
Specifieity
of
ITT
and
RA
in
adult
ehic:kens
in
f1oel<
infec:ted
with
M.
avium
Post
mortem
findings
!
Number
Test
Number
%
positive
I
negstive
of
birds
number
1
%
number
I
%'
ITT
32
29.6
24
I
75.0 8 25.0
108
whole
blood
RA
48
44.4
34
70.8 14 29.2
ITT
+RA
28
26.0
28 100.0 0 0
1
Table
2
Reliability
of
RA
in
c:hic:kens infec:ted
experimentally
with
M.
intraeeUulare
Post
mortem
lesions
Results
of
RA
with antigens
'.
Total
I
M.avium
I
M.
intracellu1are
number
of
number
of
birds
score
affected
-
I I
posit. negat. posit.
neglit.
0
14
0
14 6
8
100%
100%
57.5%
I
34
2 32 24
10
100%
5.2%
94.2%
70.6%
29.4%
55
II
7
2 5
4
3
100%
28.5%
71.5%
57.1%
.
42.9%
III
0
0 0 0
0
total
55
4
51
34
21
100%
7.2%
92.8%
61.8%
38.2%
•
•

•
•
67.
Table
3
Results
of
ITT
and
RA
in
ftoc:ks'
showing
paraaUergic:
reac:tion&
to
avian
tubereulin
Number
of
bird.
RA
with antigens
ITT
M.
intracellulare
1
M.avium
Total
positive
I
negat.
positive
I
negat.
positive
I
negat.
I
20
1
·1
14
I
6
I
0
1
0
21
1 1
0
~
0
Table
4
R~bmty
of
whole
blood
and
blood
serum
RA
in
c:hic:keus
infec:ted
experimentally
wbit
M.
intrac:eUulare
Total
number
I
1
Blood
serum
RA
antigen
I
Whole
blood
RA
antigen
of
birds
Results
I
I
M.avium
M.
intraceUur.
M.avium
M.
intracellur.
I
I
positive
I 25
2,
17
3.7%
92.6%
7.4%
62.9%
27
negative 26
2 25
10
96.3%
7.4%
92.6%
27.1%
ITT
and
RA
provide reliable information especially for the condol
of
avian
tuberculosis by the eliinination method. Parallel examinations by
both the tests
are recommended for this purpose. RA can replace
ITT
in
the screening
of
tuberculosis
in
poultry
flocks
and other bird colonies. Its major merit is that
each bird must be handled only once.
RA
is irreplaceable
in
water fowl and all
other bird species
in
which
ITT
into wattle
is
not possible. Blood serum RA
yields more accurate results and is recommended for the examination
of
birds
kept in zoological gardens and rearing colonies
of
predatory birds.
Diagnostika aviBrni
tuberkul6zy
- mykobakteri6zy
pomocl
RKA
Pfi overovam citlivosti a specifity
antigenuM.
amum seroyaru
2~
3 k rychle
• kapkove aglutinaci (RKA) jsme zjistili, ze spolehlivost uvedene inetody se zvy-
sovala u
vysetfovane drubeze na rozdil od tuberkulinace s pokroCilosti tuberku-
16zniho
procesu. U kufic infikovanych per
os
virulentnim kmenem
M.
avium
serovar 2, v davce 1 mg kultury na kg
z.
hm. reagovalo na
aviBrni
tuberkulin
pouze 47,7
%.
Pfi RKA reagovalo pozitivne v temze obdobi pfi vysetfeni pIne
krve 61,6 % a krevniho sera 90,9 % drUbeze. V nekterych chovech
drUbde
prostjch
aviBrni
tuberku16zy byly zjisiovany reakce na
aviBrni
tuberkulin, ktere
nebyly vyvolany infekci
M.
avium. Castou pfiCinou techto reakci byly piliny
kontaminovane
M.
intracellulare serovar 8. Antigen
M.
avium bylo momo
pouZ1t
pro odliseni paraalergickjch reakci na aviarni tuberkulin. Dosdene vjsledky
s tuberkulinaci a RKA prokazaly,
Ze
antigen
M.
amum byl vhodny zejmena pro
vysetfeni v chovech, ve kterjch byla ozdravovana
drubd
nebo puici pomoci
eliminaCni
metody. V techto pfipadech doporueujeme pouZit
souCasne
tuberkuli-:
nace a RKA. Krome toho bylo mozno pouZit antigen k RKA pro plosne
vySetfen1
drUbde a
ptakU
nahrazujici tuberkulinaci. U
ptakU
v zoologickjch zahradach
• nebo v chovech
dravcU
doporueujeme k vysetfeni pouZit krevniho sera,
kterjm

68
je momo
~ajistit
pfesn~si
vjsledky. Vyhodou antigenu M. avium
kRKA
je vsak •
jeho
vetSi
specifita ve srovnant s tuberkulinacf. Vzhledem k vjskytu patogennich
kmenu M. avium, ktere
ne1ze
zafadit do referencnich serovar'li M. avium
1,2,3
doporueujeme venovat
vjberu
aviSrnich
kmeOli
k pfiprave antigenu i nadlile po-
zomost.
AH8rHOCTHK8
yY6epKyns38
- MHK068KTSPH038
nTHq
c nOMO",blO RKA
B XOAe
npoBepKH
4YBcTBHTenbHocTH H cnellHcpH4HOCTH aHTHreHa M.
avium
80pKH
.2,3
AnSI
6blCTporo
KanenbHoro
arrmOTHHHpoBaHHSI (RKA)
HaMH
6blno
YCTaHoBneHO,
4TO
HaAe>KHOCTb
AaHHoro
MeTOAa YBenH4HBa-
naCb y
HccneAyeMoH
nTHlIbl B npoTHBOBec
Ty6epKynHHH3allHH
C ypOBHeM
npOllecca
Ty6epKyne3a.
Y
KypHII,
nepopanbHO
HHCPHlI~pOBaHHbIX
BHpy-
neHTHblM WTaMMOM M. avium BapKH 2
A030H
1
Mr
KynbTYPbl
HO
1
Kr
>KH-
BOH
Maccbl
Ha aBHapHblH
Ty6epKynHH
npopearHpoBanHnHwb
47,7
%_
npH
RKA
n03HTHBHO B
TOT
>Ke
nepHOA
pearHpOBanH
npH
HccneAoBaHHH
•
KpOBH
b1,6 % H
KPOBSIHOH
CblBOPOTKH 90,9 % nTHlIbl_ B
HeKoTopblX
nTH-
qeBoAcTBax
6e3
HanH4HSI
aBviapHoro
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M. avium.
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npH4HHOH AaHHoH peaKIIHH
SlBHnHCb
onHnKH,
3apa-
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intracellulare
BapKH
8_
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M.
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napaanneprH4eCKHx
peaKIIHH Ha
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pe3ynbTaTbi
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H
RKA
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nH,
4TO
aHTHreH M. avium
npHroAeH
B oco6eHHoCTH
AnSI
HccneAoBaHHSI
Ha
nTHlIeBOACTBax, B KOTOPblX npOBoAHnH 03AOpOBneHHe nTHlIbl HnH Ky-
pHlI
MeTOAOM 3nHMHHaIlHH. B AaHHoM
cny4ae
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RKA.
nOMHMO
3Toro
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npHMeHHK K
RKA
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o6utero
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llIHH
Ty6epKynHHH3a1lHIO. Y nTHlI B
300napKax
HnH
npH
pa3BeAeHHH XHut-
HblX nTHlI
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AnSI
HccneAOBaHHSI CbIBOPOTKH,
c nOMoutblO
KQTOPOH
MO>KHO
o6ecne4HTb
60nee
T04Hbie
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M.
avtum K
RKA
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ero
60nbwaSl cnellHcpH4HOCTb
no
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naToreHHblX
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avlum,
KOTopble
Henb3S1
BKnlO4HTb B
HccneAyeMble
BapKH
M.
avium . 1, 2, 3,
peKoMeHAyeM
B
AanbHeHweM
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aBHap-
•
HblX WTaMMOB
AJ:lSI
nOAroTOBKH aHTHreHa
npHcTanbHoe
BHHMaHHe.
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