Evaluation of adenosine deaminase seric activity in the diagnosis of bovine tuberculosis

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 101(4): 391-395, June 2006
391
Evaluation of adenosine deaminase seric activity in the diagnosis
of bovine tuberculosis
Márcio Roberto Silva/*, Pedro Moacyr Pinto Coelho Mota**,
Ricardo de Miranda Henriques Leite***, Francisco Carlos Faria Lobato,
Rômulo Cerqueira Leite, Andrey Pereira Lage/+
Núcleo de Pesquisa em Saúde Animal, Departamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade
Federal de Minas Gerais, Av. Antônio Carlos 6627, 30123-970 Belo Horizonte, MG, Brasil *Embrapa Gado de Leite, Coronel
Pacheco, MG, Brasil **Laboratório Nacional Agropecuário/MG, Ministério da Agricultura, Pecuária e Abastecimento, Pedro
Leopoldo, MG, Brasil ***Empresa de Pesquisa Agropecuária da Paraíba, João Pessoa, PB, Brasil
Determination of seric levels of adenosine deaminase (ADA), an enzyme produced by monocytes/macrophages
and lymphocytes, has been used in the diagnosis of human tuberculosis (TB). In the present study, ADA seric activity
was evaluated comparatively to the comparative tuberculin test in the diagnosis of bovine tuberculosis. Two hun-
dred fifty-six cattle were classified by origin and by the comparative tuberculin test as TB-positive animals (n = 52,
from herds where the Mycobacterium bovis had previously been isolated), and TB-negative animals (n = 204, TB-
free herds). The mean ADA seric value from the TB-positive group (4.45 ± 2.33 U/L) was significantly lower (p =
0.008) than that observed in sera from the TB-negative group (6.12 ± 4.47 U/L). When animals from a herd with
clinical cases of enzootic bovine leukosis of TB-negative group were withdrawn from analysis, the mean ADA seric
values of TB-negative group (5.12 ± 3.75 U/L) was not significantly different anymore from that of the TB-positive
group (p = 0.28). There was no agreement in the diagnosis of bovine TB between comparative tuberculin test and
determination of ADA seric values, using two different cutoff points, being 6.12 U/L and 15.0 U/L, (kappa = –0.086
and kappa = –0.082, respectively). In conclusion, the determination of ADA seric activity was not a good auxiliary
test for bovine TB, because it was not able to distinguish between TB-positive and TB-negative animals.
Key words: bovine tuberculosis - cattle - diagnosis - adenosine deaminase
Bovine tuberculosis (TB) is an important zoonotic dis-
is not very sensitive, the development of more sensitive
ease in cattle caused by Mycobacterium bovis and is more
and specific diagnostic tools could improve bovine TB
frequent in developing than developed countries (Cosivi
control and eradication programs (Harboe 1990, Cousins
et al. 1998, Mota & Lobato 1998). Although the disease
et al. 1991, L’Herminez 1993, Wood & Rothel 1994, Collins
has been considered under control in many developed
1995, Thoen & Bloom 1995).
countries, its reemergence has been observed during the
The enzyme adenosine deaminase (adenosine amino
last couple of years (Collins et al. 1994). In Brazil, the
hidrolase - E.C.3.5.4.4. - ADA) is an enzyme involved in
mean prevalence of M. bovis infection in cattle was esti-
the metabolism of purines (Koehler & Benz 1962, Ammann
mated to be 1.3% in the period of 1989 to 1998 (Brasil
& Fudenberg 1980, Giusti & Galanti 1984) and is widely
2005). In the state of Minas Gerais, a survey of 22,990
distributed in lymphoid tissues (Edwards et al. 1971). ADA
animals from 1586 herds performed in 1999 showed a preva-
acts in the differentiation of lymphoid cells (Barton &
lence of M. bovis infection of 5% of the herds and 0.8% of
Goldshneider 1979, Bothamley et al. 1995), is secreted in
the animals (Belchior 2001). Herd prevalence increases to
biological fluids during the cellular immune response
15% in herds with higher technological management level
against intracellular pathogens (L’Herminez 1993), but can
(Belchior 2001).
also be increased in other pathological processes
The method recommended by the World Animal Health
(Chalhoub et al. 1996). The genetic deficiency of ADA
Organization (OIE) (Palmer 2004) and worldwide accepted
production leads to a syndrome characterized by reduc-
for the diagnosis of bovine TB is the tuberculin test
tion of T cell population in humans (Clark 1995).
(Langenegger et al. 1981, Monaghan et al. 1994, O’Reilly
In human patients, the determination of ADA activity
1995, Palmer 2004). However, because tuberculin testing
in several biological fluids is used as an auxiliary diag-
nostic tool for TB, mainly for extrapulmonary TB
(L’Herminez 1993, Pérez-Rodriguez & Castro 2000, Golden
& Vikram 2005), demonstrating in those clinical forms high
sensitivity and specificity (Chalhoub et al. 1996, Pérez-
Financial support: Sebrae MG, Biokits Indústria e Comércio
Rodriguez & Castro 2000, Golden & Vikram 2005).
Ltda., FEP/MVZ Coordenação Preventiva
Rodrigues et al. (1997) evaluated ADA seric activity in
MR Silva: Capes fellowship; RMH Leite, FCF Lobato, RC
150 comparative tuberculin test (CTT)-negative animals
Leite, and AP Lage: CNPq fellowships
+
and 10 CTT-positive animals from the state of Bahia, Bra-
Corresponding author: alage@vet.ufmg.br
Received 9 January 2006
zil and ADA seric activity was significantly higher in dis-
Accepted 27 April 2006
eased cattle.

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392
392
ADA seric activity in bovine tuberculosis • Márcio Roberto Silva et al.
The aim of this study was to evaluate the ADA seric
test (Snedecor & Cochran 1980). The statistical analysis
activity comparatively to the comparative tuberculin test
were performed by Epi Info (Dean et al. 1995), Minitab
in the diagnosis of bovine TB in cattle in the state of
(Minitab for Windows, release 9.2, Minitab Inc., US) and
Minas Gerais.
WinEpiscope (Frankena et al. 1990) softwares.
MATERIALS AND METHODS
RESULTS
Animals - Two hundred fifty-six adult Holstein-Zebu
The minimum and maximum ADA seric values (U/L),
crossbred cattle from six dairy farms in Minas Gerais, Bra-
the mean values and standard deviation from TB-positive
zil, were submitted to comparative tuberculin test. Based
and TB-negative animals, as well as the global values for
on their origin and results of the CTT, animals were clas-
the two groups, are shown in Table I.
sified in two groups: (i) a TB-positive group, including 52
The mean ADA seric values for TB-positive animals
CTT-positive animals from three farms where the M. bovis
was significantly lower (P = 0.008) than that observed for
had previously been isolated from animals with clinical
TB-negative animals. Also, the highest ADA seric values
signs of tuberculosis (32 animals from Herd A, 2 from Herd
were found in TB-negative animals from two herds. When
B, and 18 from Herd C), (ii) TB-negative group, including
animals from Herd E, which had the largest percentage
204 CTT-negative animals from three tuberculosis-free
(16%) of animals with ADA seric values over 11.22 U/L
farms (5 animals from Herd D, 104 from Herd E, and 95
(the highest ADA seric values found among TB-positive
from Herd F).
animals) were withdrawn from analysis, the mean ADA
seric values between TB-negative and TB-positive cattle
Serum samples - Blood samples were collected through
did not differ significantly anymore (P = 0.28).
jugular venopunction and sera were prepared and stored
The dispersion and frequency of ADA seric values
at –20oC until tested. The time between blood collection
between TB-positive and TB-negative groups are shown
and serum storage was always shorter than 20 h. Only
in Figs 1 and 2, respectively.
hemolysis-free sera were used because ADA activity from
To study the agreement between the determination of
erythrocytes has been shown to increase ADA seric val-
ADA seric values and the CTT in the diagnosis of bovine
ues (Giusti & Galanti 1984).
TB, two cutoff points were established. One was based
CTT - The CTT was performed according to Lan-
genegger et al. (1981) using bovine and avian PPD
(Laboratório Regional de Apoio Animal, Ministério da
Agricultura, Pecuária e Abastecimento, Pedro Leopoldo,
MG, Brazil).
ADA seric activity - ADA seric activity was determined
by ADA-Kit (Biokits, Contagem, MG, Brazil), according to
the manufacturer, whose methodology is based on Giusti
and Galanti (1984), modified by Rodrigues et al. (1993).
Statistical analysis - Mean values of ADA seric activ-
ity from TB-positive and TB-negative cattle were com-
pared by Student’s t-test after logarithmic transformation
[log (ADA+1)] (Snedecor & Cochran 1980). Pearson
10
correlation coefficient was computed for data on ADA
seric activity and comparative tuberculin test (Snedecor
& Cochran 1980). The frequency and dispersion of ADA
seric activity values between TB-positive and TB-nega-
tive groups were evaluated graphically. The agreement
between ADA seric values and CTT results were evalu-
Fig. 1: adenosine deaminase (ADA) seric values (U/L) in sera from
animals found positive (52) and negative (204) by the comparative
ated by the kappa statistic (Smith 1995) and Fisher exact
tuberculin test.
TABLE I
Values of adenosine deaminase (ADA) in sera from tuberculosis (TB)-positive and TB-negative cattle by the comparative
tuberculin test
ADA seric values (U/L)
Comparative tuberculin test
Mean ± SD
Minimum
Maximum
Positive (n = 52)
4.45 c/d ± 2.33
0.00
11.22
Negative a (n = 204)
6.12 c ± 4.47
0.00
23.52
Negative b (n =100)
5.12 d ± 3.75
0.00
23.52
Total (n = 256)
5.78 ± 4.24
0.00
23.52
a: including animals from Herd E; b: excluding animals from Herd E; c: P = 0.008, d: P = 0.28

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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 101(4), June 2006
393
on the mean ADA seric value of 6.12 U/L from animals of
1993, Pérez-Rodriguez & Castro 2000, Golden & Vikram
the TB-negative group (Table II) and the other on the
2005), in areas where the prevalence of TB effusions ex-
mean ADA seric value of 15.00 U/L observed in ten TB-
ceeds other effusion causes (Burgess et al. 1995, Pérez-
free animals from the state of Bahia, Brazil (Rodrigues et
Rodriguez & Castro 2000, Golden & Vikram 2005).
al. 1997). The kappa values between ADA seric value de-
The mean seric value of ADA observed among TB-
termination and the CTT were 0.086 and –0.082 using the
negative animals in the present study was lower than that
6.12 U/L and the 15.00 U/L cutoff points, respectively
reported by Rodrigues et al. (1997), but similar to that
(Tables II, III). The Fisher exact test did not demonstrate
observed by Yasuda et al. (1996). These differences can
any association between ADA seric activity and the CTT
be related to the presence of other pathogens or uncon-
(P > 0.05).
trolled pathologies, as enzootic bovine leukosis, as has
The Pearson correlation coefficient between the ADA
been described by Yasuda et al. (1996). Surpringly, the
seric activity and the CTT was low (r = 0.129).
mean ADA seric value observed here was higher in the
TB-negative than in the TB-positive group, and this was
DISCUSSION
mainly due to the high frequency (16.34%) of animals from
The increase of ADA seric activity in biological fluids
Herd E that showed ADA seric values over 11.22 U/L,
has been used, with good sensitivity and specificity, as
similar to the highest value found among TB-positive
an auxiliary diagnostic tool in human TB, mainly for
animals. When cattle from Herd E were removed from the
extrapulmonary TB as in patients with pleural effusions,
statistical analysis, no significant difference was found
pericardial effusions, meningitis, and ascitis (L’Herminez
between the two groups. The high ADA seric values
Fig. 2: frequency of adenosine deaminase (ADA) seric values (U/L) from cattle found positive (52) and negative (204) by the comparative
tuberculin test.
TABLE II
TABLE III
Agreement of adenosine deaminase seric (sADA) values, with
Agreement of adenosine deaminase seric (sADA) values, with
a cutoff of 6.12 U/L, and comparative tuberculin test in the
a cutoff of 15.00 U/L, and comparative tuberculin test in the
diagnosis of bovine tuberculosis a
diagnosis of bovine tuberculosis a
Comparative tuberculin test
Comparative tuberculin test
sADA
Positive b
Negative c
Total
sADA
Positive b
Negative c
Total
Positive d
15
82
97
Positive d
0
12
12
Negative e
37
122
159
Negative e
52
192
244
Total
52
204
256
Total
52
204
256
a: Kappa = – 0.086; b: animals positive to the comparative
a: Kappa = – 0.082; b: animals positive to the comparative
tuberculin test, c: animals negative to the comparative tuberculin
tuberculin test; c: animals negative to the comparative tuberculin
test; d: animals positive to ADA (≥ 6.12 U/L); e: animals negative
test; d: animals positive to ADA (≥ 15.0 U/L); e: animals negative
to ADA (< 6.12 U/L); P > 0.05.
to ADA (< 15.0 U/L); P > 0.05.

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394
394
ADA seric activity in bovine tuberculosis • Márcio Roberto Silva et al.
found in animals from Herd E could be due to infection by
side the other (Figs 1, 2), making it almost impossible to
agents other M. bovis, such as the bovine leukemia virus
establish a cutoff to separate the two populations and
(BLV). Indeed, clinical signs of enzootic bovine leukosis,
develop an ADA based test with any diagnostic or epide-
confirmed by histopathology, were detected in some ani-
miologic meaning. In parallel, analysis of ADA seric val-
mals from Herd E. According to Yasuda et al. (1996), ani-
ues and CTT, measured by the kappa statistics, using the
mals with clinical signs of enzootic bovine leukosis have
two cutoff points showed a complete lack of agreement
high levels of ADA seric activity.
between the two tests.
Although elevated ADA seric levels were reported from
The use of ADA seric activity as a diagnostic tool for
human TB patients (Lakshimi et al. 1992, Rodrigues et al.
bovine TB becomes even more unlikely because, similar
1993, Orphanidou et al. 1996), measurement of this enzy-
to what happens in man (Chalhoub et al. 1996), other pa-
matic activity had medium to low sensitivity compared to
thologies such as the clinical presentation of bovine en-
ADA determination in pleural fluid (Lakshmi et al. 1992,
zootic leukosis could also increase ADA seric activity
Orphanidou et al. 1996). Furthermore, Orphanidou et al.
(Yasuda et al. 1996); the latter disease is widespread in
(1996) could not find any correlation between ADA activ-
Brazil (Leite et al. 2001).
ity in serum and in pleura. In the present study, the lack of
In conclusion, due to the impossibility of establishing
a significant difference in ADA seric values between TB-
a meaningful cutoff of ADA seric values for separation of
positive and TB-negative cattle could be due to the dif-
TB-positive and TB-negative animals, as compared to the
ference between the production of ADA isoenzymes dur-
CTT, this test should not be used as an auxiliary test in
ing immune responses in unlike species to diverse stimu-
the diagnosis of bovine TB.
lus and to the clinical status of TB-positive animals.
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