Abstract Book of EAVLD2012 - eavld congress 2012

S3 - P - 20
INTERFERON-GAMMA ASSAY TO DIAGNOSE Mycobacterium bovis INFECTION IN PIGS
Michele Pesciaroli 1 , Piera Mazzone 2 , Cinzia Marianelli 1 , Sara Corneli 2 , Miriam Russo 3 , Vincenzo Aronica 3 , Michele
Fiasconaro 3 , Massimo Biagetti 2 , Marcella Ciullo 2 , Monica Cagiola 2 , Paolo Pasquali 1 and Vincenzo Di Marco 3
1 Istituto Superiore di Sanità, Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare, Italy,
2 Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Italy, 3 Istituto Zooprofilattico Sperimentale della Sicilia,Italy
Mycobacterium bovis, pig, γ-IFN test
Introduction
Different species of animals are susceptible to Mycobacterium
bovis (MB), the causative agent of bovine tuberculosis (bTB) and
may represent both "reservoir" of infection or "spillover host".
Swine, until a few years ago, was considered only a spillover host
(1). In areas where MB is still present in cattle which share the
pasture with the pig population, the marginal role attributed to the
pig in the maintenance of infection of MB is strongly questioned.
When high rates of infection are observed also in pigs, this
species may play the role of true "reservoir" (2). The diagnostic
procedures used to identify in vivo MB infected cattle, including
skin test (IDT) and γ-interferon (γ-IFN) test, are also adopted in
other animal species (3,4) but their application in pigs has not
been completely evaluated. The aim of this study was to
calculate the sensitivity of the γ-IFN test in pigs infected with MB,
compared with the results of post-mortem diagnostic tests
(pathological examination and culture).
Materials & methods
Animals: The Black Sicilian Pig is a traditional Italian breed native
of the Nebrodi Park (Sicily). These pigs are usually reared in freerange
farms and share the pasture with cattle. In this area the
prevalence of TB in cattle ranges from 5.36% to 8.58% (data
2010 Italian Ministry of Health). For the study, 100 Blacks Sicilian
Pigs, coming from farms with bTB positive cases (2), aging from
12 to 24 months, were randomly sampled at the abattoir of Mirto
(Sicily). In addition, 15 healthy pigs from tuberculosis-free herds
were sampled at the same abattoir and used to set up the
method and as negative controls.
Protocol and dose response curves of IFN-γ: Samples of whole
blood of the 15 healthy pigs were stimulated with 5μl/ml of
Pokeweed mitogen (PWM) (Sigma-Aldrich, MS, USA) and 5μl/ml
of "Phosphate Buffered Saline" (0.01M PBS, pH 7.2). The
production of γ-IFN by lymphocytes was evaluated using a
sandwich ELISA (Porcine IFN-gamma Quantikine ELISA Kit,
R&D Systems, Mn, USA) on the supernatant collected after 24
hours of incubation at 37 °C at 5% CO 2 . The stimulation with PBS
did not induce any production of IFN-γ; on the contrary, the blood
samples stimulated with PWM, showed an average production of
γ-IFN of 2633 µg/ml, with a standard deviation of 688.2 µg/ml.
The samples stimulated with PWM, with a value below 1250
µg/ml (calculated as mean - 2 SD), were excluded from the
analysis because considered unresponsive.
γ-IFN test: Heparinized blood samples, taken at the
slaughterhouse, were dispensed in aliquots of 1.5 ml in 24-well
plates (Costar, Corning Incorporated, Corning, NY, USA). The
lymphocyte stimulation was performed in duplicate: PBS, for the
evaluation of the basal value of γ-IFN (N); PWM (5 μl/ml); bovine
PPD (10μg/ml); avian PPD (10μg/ml). The PPDs (purified protein
derivatives), used in the experiment, were both produced by the
Istituto Zooprofilattico dell’Umbria e delle Marche (IZSUM),
according to the official protocols (EC Regulation N o 1226/2002).
The values obtained from each sample, expressed in units of
Optical Density (OD), were interpreted as reported below.
Table 1. Possible outcomes following lymphocytes stimulation
with avian and bovine PPDs (OD 450 nm )
AOD & BOD < 2N
Negative
AOD or BOD > 2N
AOD 2 N Positive for M. avium
BOD 2 N Positive for M. bovis
BOD/AOD 0,9 Positive for M. avium
AOD & BOD > 2N BOD/AOD 1,1 Positive for M. bovis
0.9 BOD/AOD 1,1 Doubt
N = baseline value (value obtained with PBS only)
AOD = value obtained after stimulation with avian PPD
BOD = value obtained after stimulation with bovine PPD
Pathological examination (EAP): The macroscopic examination
was conducted at the slaughterhouse in accordance with
conventional inspection methods. Samples of lymph nodes (LN)
from the head (mandibular, retropharyngeal, parotid LN), the
thorax (bronchial LN), and the abdomen (hepatic, gastric,
intestinal LN) were also collected from all the pigs.
Microbiology: Bacterial culture for MB was performed according
to the Italian Ministerial Decree (DM 592/1995) on each lymph
node. Suspect colonies, testing positive for acid fast staining,
were further identified through bio-molecular techniques (3).
Results
Of the 100 pigs involved in the study, 2 subjects did not respond
to stimulation with PWM; these animals were excluded from the
final evaluation. EAP and culture identified respectively 26
(26,5% ) and 19 (19,4%) positive pigs . γ–IFN assay identified 22
positive pigs among the 26 with tubercular lesions and 15 among
the 19 with positive bacterial culture (Table 2). The sensitivity of
the test, assuming as gold standard the traditional tests, resulted
84,6% (95% CI 52.6-100%) and 78,9% (95% CI 44.8-100%)
respectively.
Table 2: Comparison between the results obtained in γ-IFN test,
EAP and culture for M. bovis. P: Positive N: Negative.
Lesion
Culture for M. bovis
P N P N
P 22 4 15 11
γ- IFN
N 4 68 4 68
Total 26 72 19 79
Discussion & conclusions
Our results demonstrate the circulation, in the Nebrodi Park, of
MB also in the swine population that inhabits this area (2),
supporting the hypothesis of an epidemiological role of the Black
Sicilian pig as a significant reservoir of infection. Therefore, the
implementation of future control strategies also in this species is
needed. In this perspective, γ-IFN test could represent a good
tool for intra vitam diagnosis of bTB in pigs. The study results
show, in general, an acceptable level of agreement between
traditional diagnostic tests and γ-IFN test. In particular, as shown
in Table 2, there is a better agreement with the presence of
tubercular lesions rather than with the MB isolation. This feature
can be explained with a generally low sensitivity of the cultural
test (4). Animals with positive reaction at γ-IFN test showing
neither macroscopic lesions nor MB infections could be assumed
as false positive. However, they could also be ascribed to the
capacity of this immunoassay to identify recently infected
subjects with still unapparent lesions at least during the routine
post-mortem inspection. These preliminary results encourage us
to plan future research to optimize γ-IFN test performance in
swine. This study was founded by the Research Project of the
Seventh Framework Program “Strategies for the eradication of
bovine tuberculosis” (Contract Number 212414 – Acronyms TB-
STEP).
References
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