Download In vivo and in vitro diagnosis of

Rev. sci. tech. Off. int. Epiz., 2001, 20 (1), 304-324
In vivo and in vitro diagnosis of
Mycobacterium
bovis infection
L.G. Adams
Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A & M University, and the Texas
Agricultural Experiment Station, College Station, Texas 77843-4467, United States of America
Summary
T h e intradermal delayed type hypersensitivity ( D T H ) skin test, using purified
protein derivative from culture of M. bovis or M. avium, is the most frequently used
test for diagnosis of tuberculosis or detection of M. bovis infection in cattle. Many
improvements have been made to the original tuberculin test, and molecular
approaches to identify and clone antigens may lead to improved specificity and
sensitivity of D T H skin tests. Recent a d v a n c e s in t e c h n o l o g y have allowed the
development of n e w in vitro techniques, s u c h as antibody-based, cell-mediated
immunity-based and nucleic acid-based diagnostics, w h i c h allow more rapid
diagnosis than bacteriological culture. T h e choice of diagnostic technique should
consider both the population being investigated (e.g. apparently healthy animals
or a herd w i t h a high prevalence of clinical infection) and the aim of the testing
(e.g. the screening of healthy animals or confirmation of infection in animals
strongly suspected to be infected). M o r e o v e r , any evaluation of a diagnostic test
must use a carefully selected control population w h i c h is representative of the
population to be tested in terms of relative proportions of infected and
non-infected animals.
Keywords
Cattle - Diagnosis - Mycobacterium bovis - Purified protein derivative - Tuberculin Tuberculosis.
Introduction
The increasing pressures of modern transportation and trade
are creating a requirement for rapid, simple, on-the-farm
diagnostic tests for Mycobacterium bovis infection, in addition
to mass-scale surveillance testing procedures for the infection
in cattle and wildlife populations. During the past century, the
tuberculin skin lest has been the most frequently used
diagnostic test world-wide, approved for use as the primary
screening test in most countries for on-the-farm diagnosis of
M. bovis infection of cattle. While several improvements have
been made to the original intradermal tuberculin test of
Robert Koch, the tuberculin skin test has some disadvantages
for use in detecting exposed and infected cattle and wildlife,
particularly individuals. Comprehensive reviews by Francis
et al. (46), Pritchard (106), Wood and Rothel ( 1 4 8 ) and
Martin et al. (81) have focused on advances in technology and
enhancing the understanding of the strengths and weaknesses
of available diagnostic methods, particularly classical
tuberculin tests. As a consequence of identifying some of the
disadvantages of the tuberculin skin test, a resurgence of
research for the development of improved tuberculosis
diagnostic tests has occurred. The aim of this research has
been to enhance sensitivity and differential specificity, ease of
use, adaptability to automation, computerised interpretation
and reporting for individuals, herds and populations and to
reduce the" cost. The ideal tuberculosis screening test should
correctly identify animals infected with M. bovis while
avoiding the detection of non-infected animals. Thus, this
review will address recent advances that provide alternative
rationales and methods to overcome the principal obstacles to
improving in vivo and in vitro diagnosis of M. bovis infection
and tuberculosis in cattle and wildlife.
305
Rev. sci. tech. Off. int. Epiz., 20 (1)
Approaches for evaluation of
diagnostic tests for
Mycobacterium bovis infection
in cattle
Mycobacterium bovis is an obligate pathogen: the presence of
M. bovis in an animal therefore defines the state of infection.
Detection of such animals or populations (herds) is the object
of diagnostic tests and is vital to the control and eradication of
bovine tuberculosis.
Diagnostic tests may be divided into surrogate and
pathognomonic categories. Surrogate tests are those that
detect a signal which indirectly indicates M. bovis infection,
whereas pathognomonic tests are those that directly detect the
M. bovis organism itself and thus by definition are 1 0 0 %
specific.
The three basic purposes of diagnostic tests are infection
discovery, confirmation and exclusion ( 4 0 ) . Discovery tests
are used for screening apparently healthy livestock
populations to detect M. bovis-infected animals. A high
sensitivity is desired in such assays and modest false-positive
rates can be tolerated, since the discovery test is usually
followed by a confirmatory test. Confirmatory tests are used
when M. bovis infection is strongly suspected. Confirmatory
tests must have a specificity approaching 1 0 0 % , but lower
sensitivity is acceptable. Exclusionary tests are used to rule out
the presence of M. bovis infection when suspected. Thus,
exclusionary tests must have a sensitivity approaching 1 0 0 % .
Exclusionary tests are usually too expensive for discovery
purposes. Some diagnostic tests may be useful for more than
one of these purposes.
Evaluation of diagnostic tests for chronic infectious diseases
such as bovine tuberculosis is more challenging than for acute
diseases. A thorough understanding of the pathobiology of the
disease is essential, since different signals of the infection are
produced at different phases of the infection. As a result, a
particular test may perform better during one phase than
during another. Stringent application of case definitions for
truly infected and non-infected animals and animal
populations is critical to produce a fair and objective
evaluation of assay sensitivity and specificity. Equally
important is the spectrum of infection severity among the
infected animals tested. In addition, the impact of within- and
among-herd infection prevalence on the positive and negative
predictive values of the tests must be appreciated. For assays
that are quantitative, pre-test and post-test likelihood ratios
for different levels of test signal can be used to enhance the
utility of information provided by diagnostic assays. The fact
that tests can be evaluated either for individual animals or the
herd level further adds to the challenge.
A detailed description of these clinical epidemiology concepts
is beyond the scope of this review. The estimation of the
sensitivity and specificity of diagnostic tests for bovine
tuberculosis is neither simple nor sufficient. For more
thorough explanations of the methods' of evaluating
diagnostic test performance, the reader is referred to original
research publications and reviews by Yerushalmy (150),
Feinstein (40), Martin (79), Martin et al. (80, 8 1 ) and Sackett
(121).
In vivo diagnosis
Tuberculin intradermal delayed type
hypersensitivity
The tuberculin delayed type hypersensitivity (DTH) skin test,
various forms of which exist for bovine and human
tuberculosis, may be one of the oldest and the most widely
used in vivo diagnostic tests. The tuberculin test is a surrogate
test that is entirely dependent upon the appropriate
cell-mediated immunological response of the host during the
various phases of disease pathogenesis. The strains of M. bovis
used and the production and quality control of tuberculin
have been reviewed extensively (56, 1 3 2 ) , as well as the
evaluation of tuberculin preparations and injection sites and
procedures in livestock species, especially cattle (29, 3 8 , 4 6 ,
9 8 , 9 9 ) . Tuberculins are complex mixtures of soluble antigens
(34, 4 8 ) produced by mycobacteria (M. bovis or M. avium)
grown as floating cultures in synthetic liquid media, harvested
after the mycobacteria are heat killed and removed by
nitration followed by heat concentration (old tuberculin) (64)
or chemical fractionation (purified protein derivative [PPD])
(125). Purified protein derivative is the antigen of choice for
intradermal injection at various doses (46, 5 2 , 9 8 ) . The PPD
from culture of either M. bovis (PPD-B) or M. avium (PPD-A)
is used at a 100 µl volume, either for the caudal fold, the single
intradermal cervical or the intradermal comparative cervical
test. Increases in skin thickness due to DTH-are detected by
manual palpation and/or measurement with calipers at 72 h
post injection. The caudal fold test for cattle, the single
cervical test for deer and the comparative cervical tests require
the handling of animals at least twice at a 72 h interval, do not
permit repeat testing of the same animal within sixty days,
and are more effective when used on a herd rather than
individual animal basis.
In cattle vaccinated with bacillus Calmette-Guérin (BCG) or
culture-positive for M. bovis, PPD-B induces messenger
ribonucleic acid (mRNA) expression of gamma interferon
(IFNy), interleukin (IL)-2, IL-4, IL-10 and tumour necrosis
factor-alpha (TNF-a) at the injection site at 72 h post injection
(94). Publications assessing the diagnostic efficiency of the
various skin tests report that the estimated sensitivity ranges
from 7 0 % to 9 0 % , while the estimated specificity varies from
7 5 % to 9 9 . 8 % ( 2 9 , 4 6 , 6 6 , 8 1 , 1 1 8 , 143). The caudal fold test
was reported to have a sensitivity of 7 2 % with a specificity of
Rev. sci. tech. Off. int. Epiz., 20 (1)
306
up to 9 8 . 8 % ; the single intradermal cervical test had a
sensitivity of 9 1 . 2 % with a specificity of 7 5 . 5 % . The
comparative cervical test with PPD-B and PPD-A had a
sensitivity of 6 8 . 6 % - 9 5 % with a specificity of 8 8 . 8 % - 9 9 . 9 %
(29, 4 6 , 1 1 8 , 1 4 9 ) , depending upon the circumstances in
each country, the interpretation parameters, quality and dose
of tuberculins, timing of prior tuberculin test, cross-reacting
environmental mycobacteria and other organisms, temporal
variations and chronicity of infections in populations, timing
of testing relative to calving, and the skill of the applicator.
A direct relationship exists between the predictive value of a
positive test and the prevalence of M. bovis infection, while the
predictive value of a negative test and the prevalence of
infection are inversely related. The rate of false-negatives is
influenced by time since exposure to field strain,
suppressive/anergic regulation of the immune response,
injection of insufficient tuberculin, use of tuberculins of
reduced potency, immunosuppression during the early
postpartum, desensitisation following tuberculin tests and
variability among observers ( 3 5 , 4 6 , 8 4 ) . Dietary restriction
(36) and the use of oil-based foot and mouth disease vaccines
were unrelated to false-negative results ( 8 7 ) . Using the
example of Martin e£ al. (81) to further explain the variability
of test performance in different cattle populations, applying a
tuberculin test with 8 5 % sensitivity and 9 9 % specificity with a
5% prevalence rate, the predictive value of a positive test is
81.7%. However, when the prevalence is less than 1%, more
than 5 0 % of the positive results would be expected to be
false-positive, thus the apparent prevalence is usually higher
than the true prevalence.
To address the issue of false positive tuberculin reactions in
humans, individual recombinant proteins and cocktails of
cloned recombinant tuberculo-proteins were evaluated for the
ability to elicit strong and specific DTH in PPD-positive
guinea-pigs or humans (65, 77, 1 1 4 ) . Despite protein
purification
and molecular cloning, no
individual
M. tuberculosis proteins or cocktails of recombinant proteins
were found to eliminate the specificity problems of PPD or to
elicit DTH-like responses to PPD. These findings concur with
those reviewed elsewhere associated with the genetic diversity
of the immune response of outbred cattle populations,
especially when highly purified tuberculo-protein antigens are
used (148). However, Coler et al. (18) recently cloned and
expressed a M. tuberculosis 9 kDa protein, DPPD, which elicits
a strong and specific DTH having no cross-reactivity with
other pathogenic or environmental mycobacteria, thus
providing the first putative specific and sensitive alternative
tuberculin reagent for the diagnosis of tuberculosis. The
DPPD gene has so far been found in M. tuberculosis and
M. bovis-BCG, but not in M. avium, M.
kansasii,
M. scrofulaceum, M. fortuitum, M. chelonae, M. gordonae,
M. terrae, M. smegmatis or M. vaccae ( 1 8 ) , which may
represent a major advantage for differential diagnosis and
reduction of false positives. Thus, modem molecular
approaches to identify and clone DTH diagnostic antigens
may provide new reagents for detecting M. bovis-infected
livestock with equal or greater sensitivity and specificity than
current PPD antigens, perhaps even allowing vaccination of
animals against tuberculosis while discriminating between
M. bovis-infected and vaccinated populations.
In vitro diagnosis
Direct diagnosis via morphology, histochemistry,
immunohistochemistry or polymerase chain
reaction amplification and/or hybridisation
Presumptive diagnosis of bovine tuberculosis via DTH
response to PPD, clinical history, herd history, clinical
findings and gross necropsy findings is significantly
reinforced and/or confirmed by histopathological results,
histochemical and immunohistochemical staining and
especially by in situ hydridisation with M. bovis-specific
probes. The gross and histopathological findings and the
distribution of tuberculous lesions are directly influenced by
the route, dose and virulence of the tubercle bacillus, the
susceptibility of the host animal and the time after challenge.
The pathogenesis (39, 9 2 ) and detailed descriptions of the
gross pathology and histopathology of tuberculosis have been
extensively described for naturally occurring (6, 13, 2 6 , 39,
90, 1 0 9 , 137, 144) and experimentally induced tuberculosis
(9, 10, 12, 14, 8 9 , 1 0 0 ) . However, gross lesion inspections
not infrequently fail to detect minor or invisible lesions
(particularly in the upper respiratory tract and lymph nodes of
the head) in skin-test negative or positive animals that are
subsequently found to be M. bovis culture-positive (6, 2 6 , 28,
9 1 , 1 4 4 ) , causing concern regarding the efficiency of accurate
diagnoses and the associated implications for tuberculosis
control programmes.
Thorough gross and histological examination of six pairs of
lymph nodes (mediastinal, medial retropharyngeal, bronchial,
parotid, prescapular and prefemoral) together with the
mesenteric nodes and lungs will detect 9 5 % of cattle with
gross tuberculous lesions (6, 2 6 ) . Following the detection of
granulomatous lesions compatible with tuberculosis, the
Ziehl-Neelsen (76) or the auramine O (85) acid-fast stains
may be applied to impression smears or histosections to
identify acid-fast bacilli. If the bacilli are confirmed to be acid
fast, then immunohistochemical staining (14, 5 4 ) with
antibodies specific to M. tuberculosis complex (not M. bovisspecific) and/or polymerase chain reaction (PCR) (67, 83,
122, 142), using either a 2 4 8 base-pair (bp) segment of
IS1081 or a 123 bp segment of IS6110, may be performed to
specifically identify M. tuberculosis complex organisms in
tissues or paraffin-embedded histological sections. (For
infection of cattle, when a M. tuberculosis complex organism is
identified, the likelihood that it is M. bovis is extremely high.)
Immunohistochemical staining of tissue sections had a
sensitivity equal to or greater than Ziehl-Neelsen staining (53)
on tuberculous tissues and provided a more specific M. bovis
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Rev. sci. tech. Off. int. Epiz., 20 (1)
diagnosis. Tests based on PCR performed on formalin-fixed
paraffin-embedded sections with typical tuberculous lesions
and acid-fast bacilli were 9 3 % positive ( 8 3 ) , whereas
PCR-based tests on fresh tissues were 9 1 % positive ( 1 4 2 ) .
However, the PCR results were highly specific for
M. tuberculosis complex mycobacteria and were obtained
more rapidly than bacteriological culture (three to four weeks
faster). Lastly, M. bovis has been detected by a
deoxyribonucleic acid (DNA) oligonucleotide probe/PCR
assay directly from bovine blood using general A l / B l primers
for spacer regions of the 16S and 2 3 S ribosomal RNA genes
specific for the M. tuberculosis complex and hybridised with
an oligonucleotide probe specific to M. tuberculosis complex
with a claimed analytical sensitivity of ten organisms in
100 µl of blood (5). Immunohistochemical and PCR-based
procedures offer rapid, specific, confirmatory tests for M. bovis
infection, for instance on sections or tissues with typical
tuberculous lesions and acid-fast bacilli, or when herd history,
tuberculin test results and necropsy findings are available.
Bacteriological culture
The success of primary isolation of M. bovis from clinical
specimens is influenced by the culture medium employed, the
decontamination procedure and incubation conditions ( 2 6 ) .
Decontamination procedures for the primary tissues may
affect the recovery of the mycobacteria significantly, thus
these
procedures
must
be
followed
carefully.
Decontamination has been achieved without adverse effects
on recovery using 0 . 0 7 5 % hexadecylpyridinium chloride
(26). Standard bacteriological procedures are well known
(101, 134, 1 3 7 ) . Most laboratories recommend the use of
both an agar-based medium (Middlebrook 7H11) and an
egg-based medium (Stonebrink or Löwenstein-Jensen with
pyruvate) for primary isolation. Modified Middlebrook 7H11
medium (140), B 8 3 medium (30) and radiometric detection
(88) improved the recovery rate and speed of culture when
used in conjunction with standard decontamination and
concentration procedures. Identification of mycobacterial
isolates may be obtained by standard procedures including
acid-fast staining, colony morphology, and a panel of
biochemical tests ( 1 1 3 ) , such as susceptibility to 2-thiophene
carboxylic acid hydrazine (TCH) and isoniazid. These
procedures require an additional three to four weeks. The use
of monoclonal antibodies (27) or molecular biology
techniques reviewed below allow definitive identification of
M. bovis and DNA fingerprinting of the isolate to be
performed in one day.
Antibody-based diagnostics
A sensitive and specific serological test to detect antibodies to
M. bovis antigens in domestic livestock and sylvatic
populations exposed to tuberculosis would be very useful to
replace or supplement the intradermal test. In general,
antibody production by infected individuals is variable,
largely undetectable during the early subclinical stages of
tuberculosis, and usually occurs during the advanced stages of
the disease when cell-mediated immunity tests may be
negative due to anergy. The spectrum of purified and complex
crude antigens identified and the use of these antigens in a
variety of antibody detection procedures, mostly based on the
enzyme-linked immunosorbent assay (ELISA), are listed in
Table I with comments on the sensitivity and specificity for
experimentally and naturally infected cattle (reactivity).
The sensitivities and specificities of virtually all the
tuberculosis serological tests, including the indirect and
competitive ELISA and Western blot, are relatively poor. This
is probably due to the high degree of polymorphism in the
antigen recognition, variable kinetics of the antibody
response, possible M. bovis heterogeneity, and variable rate of
disease progression of tuberculosis from animal to animal.
Additionally, high rates of false positive serological tests are
observed
due
to
extensive cross-reactivity
among
mycobacterial species. Thus, given the diversity in antigen
recognition among cattle, with the possible exception of the
memory immunoglobulin (Ig) G recombinant MPB70 ELISA
(73) before and after skin testing, serological tests are best
used
to complement
cell-mediated
immunity-based
diagnostic tests.
1
Cell-mediated immunity-based diagnostics
Given that the predominant
immune response to
mycobacterial infections in cattle is cellular rather than
humoral, development of in vitro tests to replace or
complement the intradermal skin test would greatly facilitate
control and eradication programmes. Table II documents
studies to develop and evaluate such assays in cattle
populations experimentally and naturally infected with
M. bovis. Although the intradermal skin test can be useful for
the detection of infected herds of cattle, the problem of
false-positive test results in individual cattle associated with
exposure to atypical mycobacteria highlights the need for tests
with higher specificity. The developments range from the
early lymphocyte blastogenic assays to the detection of
specific lymphokines from specific T cells stimulated with
crude, purified and recombinant antigens, or synthetic
peptides (Table II). Thus, extensive progress has been made in
the understanding of the diversity of host cellular immune
responses to mycobacterial antigens, yet much remains to be
accomplished.
The comparative (PPD-B versus PPD-A) sandwich bovine
IFNy ELISA for M. bovis developed by W o o d et al. ( 1 4 5 ) and
Rothel et al. ( 1 2 0 ) is the in vitro cell-mediated immune
diagnostic test that competes favourably with the skin test for
both sensitivity and specificity. The IFNy ELISA has been
evaluated world-wide and found to be almost always useful as
a complement to skin-testing in most countries and, under
some conditions, suitable for use as a primary tuberculosis
test. Adaptations and refinements of the sandwich bovine
IFNy ELISA concept have allowed investigators to explore the
spectrum of bovine host cell-mediated immune responses of
T cell subsets to a wide variety of crude and highly purified,
recombinant or synthetic antigens with specific epitopes.
Rev. sci. tech. Off. int. Epiz., 20 (1)
308
Table I
In vitro s e r o l o g i c a l d i a g n o s t i c t e s t s f o r t h e d e t e c t i o n of host a n t i b o d y r e s p o n s e s t o Mycobacterium
bovis
antigens
Reference
N a t u r e / a n t i g e n preparation
Reactivity
PPD
Standard USDA
¡ELISA: good specificity and reasonable sensitivity
136
PPD
Autoclaved T C A precipitate
¡ELISA: 90% positive in culture positive cattle,
11.1% positive in negative cattle
110
M. bovis A N 5
PPD
Phenol killed non-autoclaved
filtrate
¡ELISA: less discriminating than PPD
M. bovis BCG
PPD
Phenol killed non-autoclaved
filtrate
¡ELISA: less discriminating than PPD
M. bovis A N 5
PPD
Autoclaved T C A precipitate
M. bovis W T
Whole cell
Heat killed at 60°C, 120 min
sonicate
M. bovis A N 5
MPB70
Recombinant MPB70
M. bovis BCG
Mycoside B
2-O-methyl-a-L-rhamnopyranosyl ¡ELISA: bovine a n t i - M bovis antibodies highly reactive and
specific
M. bovis
PPD
Standard PPD Weybridge
¡ELISA: only 1/5 M. bovis infected cattle positive
57
M. bovis
Phosphatide extract
Saline emulsion of phosphatide
extract
¡ELISA: only 3/5 M bovis-infected cattle positive
57
M.
PPD
Standard PPD
¡ELISA: 39.9% positive in culture positive cattle
112
M. bovis A N 5
MBP70
Chromatofocused from culture
filtrate
¡ELISA: not specific for M. bovis, 6/19 positive from
M. bovis positive cattle
M. bovis AN5
CF
Culture filtrate
¡ELISA: 50% positive in M. bovis-infected cattle with low
specificity
Non-glycosylated MBP70
Chromatofocused from
ultrafiltrated, precipitated and
dialysed culture filtrate
¡ELISA: moderately high reactivity. T h e most highly specific,
although not completely species-specific
43,44
Glycosylated and
non-glycosylated MBP70
Chromatofocused from
ultrafiltrated, precipitated and
dialysed culture filtrate
¡ELISA: strong reaction with glycosylated MPB70, but with
high cross-reactivity
43,44
MBP64
Chromatofocused from
ultrafiltrated, precipitated and
dialysed culture filtrate
¡ELISA: strong reaction and 50% positive in M. bovis
infected cattle, somewhat species-specific, but more
cross-reactive than MPB70
MBP59
Chromatofocused from
ultrafiltrated, precipitated and
dialysed culture filtrate
¡ELISA: weakest reactivity and unstable
P32
Chromatofocused from
ultrafiltrated, precipitated and
dialysed culture filtrate
¡ELISA: weakest reactivity and. unstable
39 kDa, glycosylated
Chromatofocused from
ultrafiltrated, precipitated and
dialysed culture filtrate
¡ELISA: strong reaction, but with high cross-reactivity
M bovis A N 5
HSP65
Cell sonicate
¡ELISA: strongest reactions, but with high cross-reactivity
43,44
M. bovis A N 5
HSP70
Culture filtrate
¡ELISA: strongest reactions, but with high cross-reactivity
43,44
M. bovis BCG
MPB70
Purified from culture fluid
Protein-G-based ¡ELISA: moderately high specificity, low
sensitivity with only 3/8 positive in M. bovis-infected cattle,
anti-MPB70 response subject to immune priming by PPD or
MPB70
59
M. bovis BCG
BCG85B
Purified from culture fluid
Protein-G-based ELISA: moderate sensitivity, low specificity
59
M. bovis WT
Whole cell
Heat killed at 60°C, 120 min
IgG ¡ELISA: very low specificity (15.3%)
37
M. bovis
PPD
Standard
¡ELISA: variable antibody response, enhanced by prior PPD
injection
58
M. bovis
Phosphatide
Saline emulsion of phosphatide
extract
¡ELISA: variable antibody response, enhanced by prior PPD
injection
58
M. bovis
MPB70
Chromatofocused from
ultrafiltrated, precipitated and
dialysed culture filtrate
¡ELISA: sensitivity = 18.1 %; specificity = 96.4%
M. bovis
T90/1770WT
26 kDa
Freeze-thaw-sonicate-SDS-PAGE
Western blot: high sensitivity with 9/10 positive, may be
specific for M oow's
Organism
Antigen
M. bovis
M. bovis AN5
bovis.
M. bovis A N 5
M. bovis AN5
M. bovis A N 5
M.bovis A N 5
M. bovis A N 5
M bovis AN5
110
110
111
¡ELISA: sensitivity = 73.65%: specificity = 94.1 %
¡ELISA: 15.4% positive in negative cattle, and up to 39.5%
positive in cattle with mycobacteria other than M. bovis
Western blot: bovine antibody binding to MPB70 detected
4
107
17
42
43,44
43,44
43,44
43,44
43,44
147
97
309
Rev. sci. tech. Off. int. Epiz., 20 (1)
Table I (contd)
Organism
Antigen
Nature/antigen preparation
Reactivity
Reference
M.tow'sA N 5
66, 28 and 42 kDa
TCA precipitated, sonicated cell
extract-SDS-PAGE
Western blot: 66,28,42,80,71,23 and 17 kDa antigens in
descending order of reactivity, whereas 23,33 and 14 kDa
had frequency of detection.
Sensitivity PPD ¡ELISA = 44%; sensitivity 66 kDa Western
blot = 48%; specificity 66,28 and 42 kDa Western blot = 92%
15
M. bovis A N 5
Secreted 28 kDa
Recombinant protein
Western blot: recombinant 28 kDa antigen reacted with
serum antibodies of 8/9 M. tows-infected cattle and 1/4
non-infected cattle
15
M. bovis AN5
PPD-B
Standard, Commonwealth
Serum Laboratories
¡ELISA: absorbance index - culture positive = 4.3-4.6;
culture positive = 3.4, low sensitivity, low specificity
M. bovis T/91/1378
MBSE
M. bovis sonic extract
¡ELISA: 4/4 positive from culture positive cattle
M. bovis A N 5
M. tuberculosis
Erdman
M tuberculosis
H37Ra
Mb-cell filtrate, Mb-PPD,
Mb-MPB70, Mtb
ERD-lipoarabinomannan
and Mtb H37Ra
lipoarabinomannan
Mb-cell filtrate-heat killed cell
filtrate Mb-PPD-USDA standard
Mb-MPB70-chromatofocused
from ultrafiltrated, precipitated
and dialysed culture filtrate
Mtb ERD and MtbH37Ra
lipoarabinomannan extracts
Five antigen ¡ELISA: specificity = 56.4%; sensitivity = 65.6%.
Caudal tuberculin test: specificity = 89.2%;
sensitivity = 84.9%
47
M.bovisBCG
Cell filtrate
Concentrated ultrafiltrate
Monoclonal antibody (4C3/17) cELISA: specificity = 68%
(bison) and 85% (cattle); sensitivity = 89.5% (bison) and 85%
(cattle)
130
M. bovis A N 5
MPB70
Chromatofocused from
ultrafiltrated, precipitated and
dialysed culture filtrate
¡ELISA: specificity = 73.7% (bison) and 75% (cattle);
sensitivity = 78.9% (bison) and 65% (cattle)
130
M. tow's
PPD-B
M tow's PPD-B Central
Veterinary Laboratory,
Weybridge, United Kingdom
¡ELISA: positive results were only detected in 2/13 infected
cattle on days 21 and 42 post infection
12
M. tow's
ESAT-6,14 kDa protein,
MPT63,19 kDa protein,
MPT70,MPT64, MPT51,
MTC28, Ag85B, 38 kDa
protein, MPT32 and KatG
Recombinant antigens
¡ELISA: ESAT-6,14 kDa protein, MPT63, MPT70, MPT51 and
MPT32 detected high antibody activity in experimentally
infected cattle
78
9
104
M. bovis
PPD
Standard
¡ELISA: low specificity and low sensitivity
1
M. bovis
PPD
Standard-
cELISA: low specificity and low sensitivity
1
M. bovis
rMPB70, rMPB64, rMPB59
Purified recombinant proteins
¡ELISA: high prolonged levels of anti-MPB70 antibodies, and
especially the enhanced memory response after tuberculin
testing and class switch from lgG2 to lgG1, may offer a
procedure to determine the disease status of skin test
reactor cattle
73
M. bovis
PPD
Tecpar, Brazil
¡ELISA: sensitivity = 86.7%; specificity = 90.6% under field
conditions, based upon tuberculin reactions
74
cELISA
ELISA
¡ELISA
Ig
Mb
Mtb
: competitive ELISA
: enzyme-linked immunosorbent assay
: indirect ELISA
: immunoglobulin
: Mycobacterium bovis
: Mycobacterium tuberculosis
PPD
: purified protein derivative
PPD-B
: PPD derived from M. bovis culture
SDS-PAGE : sodium dodecyl sulphate Polyacrylamide gel electrophoresis
TCA
: trichloracetic acid
USDA
: United States Department of Agriculture
Clearly, this research has furthered the understanding of the
complexity and diversity of the host immune response.
However, specific mycobacterial antigens and peptide
epitopes from the antigens studied thus far seem to be of
limited practical value in cell-mediated immunity tests, due to
the diversity of the immune recognition of these epitopes by
outbred cattle ( 1 0 4 , 1 0 5 , 1 4 1 ) . A . dynamic antigenic
repertoire, particularly one containing low molecular weight
secreted antigens may improve in vitro cell-mediated
immunity assays, particularly during early stages of infection.
These early-stage secreted, more promiscuous antigens
potentially offer a new approach for the diagnosis of bovine
tuberculosis ( 1 0 3 ) .
N u c l e i c acid-based diagnostics
Since M. bovis has the widest host range of the mycobacteria
in the M. tuberculosis complex, one of the important obstacles
to understanding the epidemiology of M. bovis transmission
among mammalian populations has been the inability to
differentiate or type strains based upon phenotypic
310
Rev. sci. tech. Off. int. Epiz., 20 (1)
T a b l e II
In vitro c e l l - m e d i a t e d i m m u n i t y - b a s e d d i a g n o s t i c t e s t s f o r t h e d e t e c t i o n of host c e l l u l a r r e s p o n s e s to Mycobacterium
spp. antigens
References
Organism
Antigen
N a t u r e / a n t i g e n preparation
Reactivity
M. oow's AN 5
PPD-B
Ammonium sulphate
precipitation of culture filtrate
Lymphocyte blastogenesis: stimulation index
M. bovis infected cattle, and 1.58 in controls
M. bovis AN5
PPD-B
Standard, Commonwealth
Serum Laboratories
Bioassay for bovine IFNy from PPD-stimulated whole blood
detected all cattle experimentally challenged with M. bovis
145
M. bovis AN5
PPD-B
Standard, Commonwealth
Serum Laboratories
MAb-based sandwich bovine IFNy ELISA analysis of
PPD-stimulated whole blood: highly specific, with results in
24 h and no requirement for testing animals after 3 days,
unlike tuberculin test
119
M. bovis
MPB70
Chromatofocused from
ultrafiltrated, precipitated and
dialysed culture filtrate
MAb-based sandwich bovine IFNy ELISA analysis of
MPB70-stimulated whole blood: significantly reduced
sensitivity with only slightly improved specificity
147
M. bovis AN5
PPD-B
Standard, Commonwealth
Serum Laboratories
MAb-based sandwich bovine IFNy ELISA analysis of
MPB70-stimulated whole blood: high sensitivity (93.6%) and
high specificity (96.2%)
148
M. bovis AN5
PPD-B
Standard, Commonwealth
Serum Laboratories
MAb-based sandwich bovine IFNy ELISA analysis of
PPD-stimulated whole blood: high sensitivity (76.8% to
93.6%) and high specificity (96.2% to 98.1 % )
146
M. bovis AN5
PPD-B
Standard, Commonwealth
Serum Laboratories
MAb-based sandwich bovine IFNy ELISA analysis of
PPD-stimulated whole blood: optimised by use of
heparinised blood stimulated with 20 µg/ml PPD within 8 h
of collection
120
M bovis
(T/91/1378)
MBSE and PPB
Ultrafiltered, sonicate
Standard PPD
Lymphocyte blastogenesis: stimulation index is high for both
MBSE and PPD for 270 days post infection with M. bovis
103
M. bovis
Synthetic peptides, PPD-I
PPD-A
Lymphocyte blastogenesis stimulation indices (>2.0 =
positive): MBP70-88-105 residues = 0.59-21.1,8/14 positive;
MBP70-144-163 residues = 0.54-35.1 and 19,8/14 positive;
19 kDa 1 -16 residues = 0.60-8.76,7/14 positive; 19 kDa
67-84 residues = 0.50-19.1,8/14 positive; MBB57-85-100 =
0.72-11.0,4/14 positive; PPD-A = 0.33-141.0, 6/14 positive;
PPD-B = 2.06-199.2,14/14 positive
103
M avium
MBP70-88-105,144-163
residues; 19 kDa 1-16,
67-84 residues;
MBB57-85-100; PPD-B and
PPD-A
M. bovis
PPD-B
Standard, Commonwealth
Serum Laboratories
Bovine IFNy ELISA analysis of PPD-stimulated blood
leukocytes of tuberculin negative cattle: of 98 IFNy positive,
15 were culture positive
93
M. bovis
PPD-B
Lyophilised, Biologics Unit,
ADRI, Nepean, Ontario, Canada
Lymphocyte blastogenesis of elk: sensitivity = 70%
specificity = 74%
61
M. avium
PPD-B
Lyophilised, Biologics Unit,
ADRI, Nepean, Ontario, Canada
Lymphocyte blastogenesis of elk: sensitivity = 74%
specificity = 41%
61
M. bovis
Old tuberculin
Biologies Unit, ADRI, Nepean,
Ontario, Canada
Lymphocyte blastogenesis of elk: sensitivity = 45%
specificity = 75%
61
M. avium
Old tuberculin
Biologies Unit, ADRI, Nepean,
Ontario, Canada
Lymphocyte blastogenesis of elk: sensitivity = 33%;
specificity = 74%
61
M. bovis AN5
PPD-B
Standard, Commonwealth
Serum Laboratories
MAb-based sandwich bovine IFNy ELISA analysis of
PPD-stimulated whole blood (BCG vaccinated and M. bovis
challenged animals): 19/20 IFNy positive and culture
positive
9
M. bovis AN5
PPD-B
Standard, Commonwealth
Serum Laboratories
Lymphocyte blastogenesis: BCG vaccinated and M. bovis
challenged animals: 16/20 IFNy positive and culture positive
9
M. bovis ANS
PPD-B
Standard, Commonwealth
Serum Laboratories
IL-2 bioassay: stimulation indices, culture positive = 7.5-16;
culture negative = 2.0
9
M. bovis
38 kDa 1-27, 88-107,
122-138,243-260,307-328
residues; MBSE; PPD-B;
PPD-A
Synthetic peptides, M. bovis
sonic extract, PPD-B and PPD-A,
CVL, Weybridge, UK
Lymphocyte blastogenesis stimulation indices
(>2.0 = positive): in PPD-B positive cattle 38 kDa residue
1-27 = 1.09-9.84; residue 88-107 = 0.93-15.49; residue
122-138 = 1.13-6.03; residue 243-260 = 0.87-3.07; residue
307-328 = 0.60-10.36; PPD-B = 37.6-128.2; PPD-A = 2.4-24.2;
MBSE = 34.5-211.4; bovine IFNy ELISA: only residue
122-138 positive
9
M. bovis AN5
PPD-B
Standard, Commonwealth
Serum Laboratories
MAb-based sandwich bovine IFNy ELISA analysis of
PPD-stimulated whole blood using manufacturer standard:
sensitivity = 73% positive, compared to 80.4%-84.4% for
caudal fold test in 204 culture positive cattle
9
(T/91/1378),
18.1 in
86 135
311
Rev. sci. tech. Off. int. Epiz., 20 (1)
T a b l e II ( c o n t d )
Organism
Antigen
Nature/antigen preparation
Reactivity
M. bovis (T/91/1378)
MBSE and PPB
M. bovis sonic extract, PPD-B,
CVL, Weybridge, UK
Lymphocyte blastogenesis stimulation indices (>2.0 =
positive): from early to late stages after experimental
infection, T y ò cells decreased, then increased. Ratios of
CD4:CD8 increased, then decreased
M. bovis ANB
PPD-B
Standard, Commonwealth
Serum Laboratories
MAb-based sandwich bovine IFNy ELISA analysis of
PPD-stimulated whole blood using manufacturer standard.
IM dexamethasone injection for 3 days at 10 ml containing
2 mg/ml suppressed the sensitivity significantly
49
M. bovis
PPD-B
Standard, CVL, Lelystad,
the Netherlands
Sandwich ELISA for bovine soluble IL-2 receptor: a analysis
of PPD-stimulated PBMC: 61/67 positive in culture positive
cattle, only 1/51 positive in uninfected controls
96
M. bovis
PPD-B
Standard
Comparison of RT-PCR and MAb-based sandwich ELISA for
bovine IFNy and IL-2 analysis of PPD-stimulated PBMC: both
are positive in culture positive cattle and correlate well with
tuberculin skin test
95
M. tuberculosis and
M. bovis
ST-CF, rESAT-6, PPD-B
M. tuberculosis, short-term
culture filtrate, recombinant
ESAT-6; M bovis. PPD-B, CVL,
Weybridge, UK
Sandwich bovine IFNy ELISA analysis of ST-CF fractions,
rESAT-6 and PPD-B PBMC from infected cattle: high
response to <10 kDa ST-CF antigens in early stages of
infection; responses to both low and high molecular weight
ST-CF antigens in later stages of infection; high responses
to rESAT-6 in majority of naturally infected cattle equal to
PPD-B
105
M. bovis
rMPB70, rMPB64,81-100
and 174-190 residues of
MPB70,1-20,41-60,
181-200 residues of
MPB64
Recombinant MPB70 and
MPB64, synthetic peptides of
81-100 and 174-190 residues of
MPB70, and 1-20,41-60,
181-200 residues of MPB64
Lymphocyte blastogenesis stimulation indices (>2.0 =
positive) and sandwich bovine IFNy ELISA analysis of
rMPB70 and rMPB64, synthetic peptides of 81-100 and
174-190 residues of MPB70 and 1-20,41-60,181-200
residues of MPB64 and PPD-B PBMC from infected or
immunised cattle: both high lymphoblastogenic and IFNy
responses were detected in most cattle but no one antigen
or epitope w a s recognised by all animals
71
M. bovis
rMPB59,101-110 residues
of MPB59, PPD-B
Recombinant MPB59
(fibronectin binding protein),
synthetic peptides of 101-110
residues of MPB59, M. bovis
PPD-B, CVL, Weybridge, UK
Lymphocyte blastogenesis stimulation indices (>2.0 =
positive) of M. bovis-specific bovine CD4 T cell clones and
sandwich bovine IFNy ELISA analysis of rMPB59 and
synthetic peptides of 101 -110 residues of MPB59 and
PPD-B-stimulation of whole blood from infected cattle: T cell
clones and infected cattle responded positively to MPB59,
but less than PPD-B, and 6/13 cattle responded to 101-110
residues of MPB59
72
M. bovis
PPD-B
M. bovis PPD-B, CVL,
Weybridge, UK
Sandwich bovine IFNy ELISA and lymphocyte blastogenesis
analysis of PPD-B-stimulated PBMC from infected cattle:
generally increasingly positive IFNy and lymphocyte
blastogenesis responses were detected continuously in 2/13
infected cattle at 7-42 days and at 14-42 days post infection,
respectively
12
M. bovis W T
MBSE, rMPB70 PPD-B
M. bovis sonic extract live
M. bovis, recombinant MPB70
and M. bovis PPD-B, CVL,
Weybridge, UK
Sandwich bovine IFNy ELISA and lymphocyte blastogenesis
analysis of CD8 and CD4 M. bovis T cell clones from
PPD-B-stimulated PBMC from infected cattle: live M. bovis,
MBSE and rMPB70 in descending order of stimulation
significantly induced both IFNy and lymphoblastogenic
responses of CD4 and especially endogenously processed
antigens for C D 8 T cells
70
M. bovis AN5
PPD-B
CZ Veterinaria, Spain
MAb-based sandwich bovine IFNy ELISA analysis of
PPD-B-stimulated whole blood: sensitivity = 84.9%,
compared to single intradermal cervical test = 80.2%,
sensitivity of combination of both tests = 92.9%, but with
several false negative and numerous false positive results
50
M. bovis A N 5
PPD-B
CZ Veterinaria, Spain
MAb-based sandwich bovine IFNy ELISA analysis of
PPD-B-stimulated whole blood from cattle of herds with
54.5% tuberculosis-'relativity': sensitivity = 100% with
several false positives, compared to single intradermal
cervical test = 88.3%
75
M. bovis
rMPB59, rMPB64, rMPB70,
rESAT-6, PPD-B
Recombinant MPB59, MPB64,
MPB70, ESAT-6, PPD-B,
'standard, Commonwealth
Serum Laboratories
Sandwich bovine IFNy ELISA analysis of rMPB59, rMPB64,
rMPB70, ESAT-6-stimulated whole blood from infected or
BCG immunised cattle: only ESAT-6 and PPD-B stimulated
high levels of IFNy; furthermore, only ESAT-6 stimulated a
differential response between BCG-vaccinated and
M. tows-infected cattle
Referenc es
103
11
*
312
Rev. sci. tech. Off. int. Epiz, 20 1)
T a b l e II ( c o n t d )
N a t u r e / a n t i g e n preparation
Reactivity
References
Organism
Antigen
M. bovis
rMPB64, rMPB70, rMPB83,
rESAT-6, 89-104 and
105-120 residues of
MPB64,1-16 residues of
MPB70,195-214 residues
of MPB83.1-16,17-32,
and 57-72 residues of
ESAT-6, PPD-B
Sandwich bovine I F N gELISA and lymphocyte blastogenesis
Recombinant MPB59, MPB64,
analysis of rMPB64, rMPB70, rMPB83, rESAT-6, 89-104 and
MPB70, ESAT-6, synthetic
105-120 residues of MPB64,1-16 residues of MPB70,
peptides 89-104 and 105-120
195-214 residues of MPB83,1 -16,17-32, and 57-72 residues
residues of MPB64,1-16
of ESAT-6 and PPD-B-stimulated PBMC from
residues of MPB70,195-214
residues of MPB83,1-16.17-32, M. bovis-infected or BCG-vaccinated cattle: rMPB64,
rMPB70, rMPB83 and rESAT-6 induced blastogenesis and
and 57-72 residues of ESAT-6,
I F N gsecretion only in infected cattle. Importantly, a cocktail
PPD-B, standard.
of promiscuous T cell epitopes 89-104 and 105-120 residues
Commonwealth Serum
of MPB64,195-214 residues of MPB83,1-16,17-32 and
Laboratories
57-72 residues of ESAT-6 detected responses only from
M óows-infected cattle
141
M. bovis
PPD-B
M. bovis PPD-B, CVL,
Weybridge, UK
14
M. bovis
rESAT-6, rMPB64, rMPB70, Recombinant ESAT-6, MPB64,
rMPB83, rhsp 16.1,
MPB70, MPB83, hsp 16.1,
rhsp 65, rhsp 70 and native hsp 65, hsp 70 and native
38 kDa lipoprotein and
38 kDa lipoprotein and Ag85A,
Ag85A, B and C complex,
B and C complex, PPD-B, CVL,
PPD-B
Weybridge, UK
Sandwich bovine I F N gELISA and lymphocyte blastogenesis
analysis of PPD-B-stimulated whole blood from infected
cattle: 8/9 calves were positive to I F N gELISA and 5/9
calves were positive to lymphocyte blastogenesis by 28 days
post infection, while no antibodies were detected by ¡ELISA
IL-2 bioassay, sandwich bovine I F N gand T G F bELISA and
ADRI
BCG
CVL
ELISA
hsp
¡ELISA
IFNg
Ig
IL
IM
MAb
:
:
:
:
:
:
:
:
:
:
:
Animal Diseases Research Institute
bacillus Calmette-Guérin
Central Veterinary Laboratory
enzyme-linked immunosorbent assay
heat shock protein
indirect ELISA
gamma interferon
immunoglobulin
interleukin
intramuscular
monoclonal antibody
Mb
Mtb
PBMC
PPD
PPD-A
PPD-B
RT-PCR
ST-CF
TGFB
UK
108
lymphocyte blastogenesis analysis of rESAT-6, rMPB64,
rMPB70, rMPB83, rhsp 16.1, rhsp 65, rhsp 70 and native
38 kDa lipoprotein and Ag85A, B and C complex, PPD-Bstimulated PBMC and draining lymph node cells from
M. oows-infected cattle: little difference w a s detected
between PBMC and lymph node cells; ESAT-6 and PPD-B
elicited the strongest responses of primarily CD4 and T g d
cells
: M. bovis
: M. tuberculosis
: peripheral blood mononuclear cell
: purified protein derivative
: PPD derived from M. avium culture
: PPD derived from M. bovis culture
: reverse transcriptase-polymerase chain reaction
: short-term culture filtrate
: transforming growth factor beta
: United Kingdom
characterisation. Thus, this section is a review of the
progressive development of nucleic acid based molecular
methods for this purpose. From the early DNA fingerprint
analysis by restriction endonuclease procedures by Collins
and de Lisle (19, 2 0 ) , to the restriction fragment length
polymorphism (RFLP) analysis by Thierry et al. (133),
followed later by the spoligotyping methods of van Soolingen
e£ al. (139) and Kamerbeck et al. (62), and now the multiplex
PCR procedures of Sreevatsan et al. (129), the detection and
differentiation of Mycobacterium spp. by nucleic techniques
clearly hold great promise for epidemiological investigations
at local, national and international population levels. Table III
summarises the research in domestic and sylvatic mammals
and provides a chronological and technological history of
nucleic acid-based procedures not only to detect M. bovis, but
to differentiate among members of the M. tuberculosis
complex and identify strains infecting different populations of
animals. This area of tuberculosis investigation has
experienced a tremendous growth in productive research
with direct application in laboratories on a world-wide basis.
The data in Table III provide compelling evidence for the
immediate application of these methods to naturally
occurring tuberculosis in any domestic or sylvatic mammal in
almost any setting. The evolution of procedures shows the
steady progress towards strain typing within sub-species of
Mycobacterium.
The ideal method of typing should be
inexpensive, reproducible, rapid, easily performed and have
direct application to clinical specimens. While none of the
current methods fulfill all these criteria, the improvements
achieved since the early 1990s have been remarkable. Most of
these procedures are adaptable to modem laboratories and are
useful tools for advancing understanding of the dynamics of
M. bovis transmission among animals. These techniques serve
as the basis for improved tuberculosis control and eradication
programmes at farm, regional, national and international
levels.
The broad spectrum of DNA fingerprinting methods for
detecting and differentiating Mycobacterium
spp. is fully
294
Rev. soi. tech. Off. int. Epa., 20 (1)
incidence is likely to be greater. Much of the following
information is based on the récent émergence of
M. tuberculosis in Asian éléphants in the USA (42).
Epidemiology
The récent émergence of M. tuberculosis in éléphants in North
America began with the death of two travelling circus
éléphants in 1996 (3). Between August 1996 and 3 June
2000, seventeen cases of M. tuberculosis were confirmed in
Asian éléphants in North America (42). Eléphants from eight
herds in Illinois, California, Arkansas, Missouri and Florida
were affected. Contact had previously occurred between
éléphants in five of the herds. Mycobacterium tuberculosis was
isolated from twelve éléphants ante mortem and from five
éléphants post mortem. Five distinct strains of M. tuberculosis
were identified, based on restriction fragment length
polymorphism (RFLP) of isolâtes from eleven éléphants.
From this study, the prevalence of M. tuberculosis was
estimated to be approximately 3.0% in the captive éléphant
population in North America (42).
Clinical and pathological aspects
Signs attributed to tuberculosis in éléphants are mostly
non-specific, e.g. inappetance, weight loss, reluctance to do
strenuous work and occasionally. subcutaneous ventral
oedema. More typically, premonitory ante-mortem signs are
absent. The major pathological changes in éléphants with
M. tuberculosis occur primarily in the lungs and thoracic
lymph nodes with lesser involvement of extra-thoracic sites.
In the less extensive cases, firm granulomatous nodules,
sometimes with caseous foci, are noted in the branchial
lymph nodes and pulmonary tissue (Fig. 1). Eléphants with
extensive involvement of both lungs usually die with severe
caseo-calcareous and cavitating lésions (Fig. 2). Thèse often
resuit in large pulmonary abscesses from which
Fig. 2
Advanced caseo-calcareous tuberculous pneumonia
(Bar = 5 cm)
Segment of pulmonary lobe from an Asian éléphant (index case) from the
1996 outbreak of Mycobacterium tuberculosis in the United States of
America
Photo: courtesy of Dr Hailu Kinde, California Animal Health and Food Safety
Laboratory, University of California, Davis
M. tuberculosis and opportunistic bacteria
Pseudomonas aeruginosa hâve been isolated.
such as
Characteristic histological findings include epithelioid
granulomas with some giant cell formation in the initial
lymph node and pulmonary lésions and extensive caseous
and pyogranulomatous pneumonia in the advanced forms.
Although sparsely distributed, acid-fast bacilli are more easily
found in central areas of caseation in the lungs, rather than in
the lymph nodes where they are typically rare. Branchial and
trachéal tuberculous plaques, and caseous and mucopurulent
exudate in the nasal and trunk passages hâve been noted in
both the early and late stages of tuberculosis, suggesting that
the shedding of mycobacteria may occur at any stage of the
disease.
Diagnosis and control
Fig. 1
Lung segments (afterfixation in formalin) depicting an early case of
tuberculosis with granulomas containing central caseation necrosis
from a forty-five-year-old female Asian performing éléphant from the
United States of America
(Bar = 1cm)
The larger nodules are surrounded by smaller satellite epithelioid
granulomas. Mycobacterium tuberculosis was cultured from the lésions
In 1997, a National Tuberculosis Working Group for Zoo and
Wildlife Species in the USA formulated the Guidelines for the
Control oj Tuberculosis in Eléphants (71) to specify criteria for
the testing, surveillance and treatment of éléphants for
tuberculosis. Intradermal tuberculin tests to diagnose
tuberculosis in éléphants hâve correlated poorly with
mycobacterial culture results, with high percentages of
false-negatives in culture-positive animais. Currently, the test
considered most reliable for diagnosis of tuberculosis in
éléphants is based on the culture of respiratory sécrétions
obtained by trunk lavage (or 'washes'). Stérile saline is instilled
into the nostrils and then recovered in a plastic bag. Three
samples are collected on separate days (26). Eléphant herds in
the USA are tested by this method on an annual basis or more
frequently if cases of tuberculosis are detected or if the herd is
known to hâve been previously exposed to tuberculosis. The
flow sheet in Figure 3 présents the testing schedule in détail. A
314
Rev. sci. tech. Off. int. Epiz., 20 (1)
T a b l e III ( c o n t d )
Insertional element/restriction
enzyme/gene
Reactivity
References
Organism
Method
M. bovis
RFLP
RFLP performed with DR, PGRS
probed Alu I digests; 245 bp IS6110
probe of Pvu II digests
IS6770, DR and PGRS individually identified 17,18 and 18
different RFLP types respectively from M bovis isolates from
cattle, feral deer, badgers, sheep, humans and swine, but the
combination of the 3 techniques identified 39 distinct types
adequate to indicate that the strains are not host restricted
M. bovis
PFBE
PFGE of genomic D N A digested with
Dra I, X t aI,SpeIor Vsp I
PFGE identified 53 patterns from isolates of M. bovis from cattle,
swine, deer, seals, badgers, possums and humans. Dra I w a s the
most useful, producing fewer fragments of larger size which could
be further sub-divided by Spe I. This w a s found to be highly
discriminatory for epidemiological investigations
41
PGR and
multiplex PGR
Multiplex PCR for mtp40 and IS1081;
multiplex PCR for 16S rRNA and
MPB70; PCR for IS6110
mtp40 was not detected in M. bovis, while ISIS1081,16S-rRNA,
MPB70 and IS6110 w e r e detected in all M. tow's isolates,
indicating that multiplex PCR of mtp40 and IS 1081 may be useful
for differentiating among members of the M. tuberculosis
complex. IS6110 w a s reported in M. gilvum and M. ulcerans for
the first time
68
Spoligotyping
Spacer oligonucleotide typing - PCR
amplification of spacers between
the DRs
M. bovis isolates from cattle and goats typed by spoligotying
revealed a common M. tow's caprine genotype which w a s useful
in epidemiological studies of M. tow's transmission among
humans and domestic livestock
55
PGR, subtractive
hydridisatlon
PCR of 125 bp sequence apparently
upstream of the isocitrate
dehydrogenase gene of M bovis
PCR of a 125 bp sequence was detected only in the
M. tuberculosis complex
60
M. bovis
RFLP
RFLP performed with RHS-245 bp
and LHS-867IS6110 probed Pvu II
digests
The combination of RFLP performed with RHS-245 bp and LHS-867
ISS7 70 probes successfully differentiated multiple copy IS6110
isolates and w a s useful for differentiating between caprine and
bovine M. bovis
69
M. bovis
PCR-SSCP
PCR-SSCP of a single base (C-to-G)
point mutation of the Pzase (pncA)
gene
PCR-SSCP of pncA differentiated 87 of 89 M. bovis isolates
123
M. bovis
BACTEC-spoligotyping
Spacer oligonucleotide typing - PCR
amplification of spacers between
the DRs w a s performed on BACTEC
cultures at various growth indices
Within 10 days, 90.4% and 94.2% of BACTEC culture positive
bovine samples at growth indices of =60 and =200 respectively
yielded definitive spoligotyping patterns. Spoligotypes w e r e
obtained from 84.6% of culture-positive samples. T w o
spoligotypes accounted for 80.7% of culture positive samples
117
M. bovis
Spoligoytping, RFLP
Spacer oligonucleotide typing - PCR
amplification of spacers between
the DRs and RFLP performed with DR
and PGRS probed Alu I digests;
1358 bp IS6110 probe of Pvu II
digests
IS6110, DR and PGRS identified 24 different RFLP types while
spoligotyping revealed 15 different spoligotypes among M. bovis
isolates from cattle, badgers and deer. Although RFLP typing w a s
more discriminatory, spoligotyping w a s easier to perform, more
rapid, easily documented and more suitable for large-scale
screening purposes
62,128
M. bovis
Spoligoytping, RFLP
Spacer oligonucleotide typing - PCR
amplification of spacers between
the DRs and RFLP performed with DR
and PGRS probed Alu I digests;
1358 bp IS6110 probe of Pvu II
digests
PGRS closely followed by IS6110 RFLP w a s found to be the most
sensitive method to identify polymorphisms among M. bovis
isolates from cattle, sheep, goats, cats and wild boar
3
M. bovis
RFLP
RFLP performed with DR and PGRS
probed Alu I digests; 1358 bp IS6110
probe of PvuW digests
Combined DR and PGRS-RFLP types identified 93 different
patterns which w e r e successfully used for cluster analysis and
epidemiological investigations
45
M. bovis
Spoligotyping, RFLP
Spacer oligonucleotide typing - PCR
amplification of spacers between
the DRs and RFLP performed with DR
and PGRS probed Alu I digests;
RHS-245 bp IS6110 probe of Pvu II
digests
The 273 M. bovis isolates from numerous domestic and sylvatic
animals from 4 countries yielded: 23 ISS6110 RFLP types, 35 DR
types, 77 PGRS types and 35 spoligotypes, while the combination
identified 99 different patterns which w e r e useful for cluster and
epidemiological investigation.IS06110RFLP type analysis was
adequate for strains having 3 copies, but IS6110 RFLP combined
with PGRS w a s necessary for typing isolates with less than 3
copies. Spoligotyping w a s found to be rapid but lacking in
sensitivity
33
M. bovis
RFLP, spoligotyping
IS6110-RHS RFLP of Pvu II digests,
spoligotyping, and PGRS-RFLP of
Alu I digests
For standardised recommendations for M. bovis isolates with 3 or
more copies, ISS6110-RHS RFLP was adequate. However, if the
isolate has 2 or fewer copies, spoligotyping was recommended
followed by PGRS-RFLP for maximum sensitivity in discriminating
strains
32
M. bons.
29
other species of
Mycobacterium
M.
tows
M. bovis, 19
other species of
Mycobacterium
128
315
Rev. sci. tech. Off. int. Epiz., 20 (1)
Table III ( c o n t d )
Organism
Method
M. bovis
PCR
Insertional element/restriction
enzyme/gene
Reactivity
References
PCR amplification of IS6110 from
C1 -carboxypropylbetaine processed
milk
Detection of M. bovis in bovine milk w a s enhanced to 94.1 % as
compared to 58.8% by standard glass bead processing
25
8
M. bovis
LCx
Amplification of 44 bp of
M. tuberculosis proteic antigen
5 after 37 cycles followed by
microparticle enzyme immunoassay
LCx had a sensitivity of 83.9% and specificity of 100% compared
to 85.7% sensitivity and 100% specificity of culture by BACTEC
460, resulting in a concordance of 81.1 % of LCx versus BACTEC
16
M. bovis
RFLP, spoligotyping
RFLP performed with DR and PGRS
probed Alu I digests; spoligotyping
Of 154 M. bovis isolates, mainly from South America,
spoligotyping identified 41 different types, DR and PGRS identified
42 types each, while combining spoligotyping with DR and PGRS
identified 88 types. Spoligotyping w a s recommended as the initial
step, followed by DR and PGRS RFLP analysis for cluster analysis
151
M. bovis
RFLP, (GTG) -PCR,
ERIC primers for PCR,
PCR ribotyping
IS6110 RFLP with 0.45 kb Bam HlSal I fragment performed on Pvu II
digests; primers for (GTG) , ERIC and
3' end of 16S and 5' 23S of rrn
ribotyping followed by Hae III
restriction were used for PCR
amplification
IS6110 RFLP identified 6 patterns, ERIC detected 7 patterns, and
(GTG) discerned 8 patterns, while PCR interspacer ribotyping w a s
not useful
124
M. bovis
500 bp PCR
PCR amplification with JB21-JB22
primers to produce a 500 bp probe
for hybridising Pvu II digests
100% of 121 M bovis isolates were detected
115
M. bovis
Spoligotyping, RFLP
Spoligotyping; IS6770and IS 1081
used to probe Pvu II digests
Spoligotyping identified 9 patterns, IS6110 differentiated a single
and a 5 copy pattern, while IS 7087 did not detect any differential
patterns in M. bovis isolates from 63 wild boar and 16 cattle.
Spoligotyping w a s the most useful procedure for cluster analysis
and epidemiological investigations
126
M. bovis
Spoligotyping, RFLP
Spacer oligonucleotide typing - PCR
amplification of spacers between
the DRs and RFLP performed with DR
and PGRS probed Alu I digests;
RHS-245 bp IS6110 probe of Pvu II
digests
Molecular fingerprinting of 29 M. bovis isolates demonstrated the
stability of profiles, despite persistence and spread within a single
herd during 2 decades, confirming the power for epidemiological
investigation
102
M. tuberculosis
complex
Species-specific
gyrB-PCR-RFLP
Rsa I and Taq I restriction of the PCR
product of the primer set 756-A and
1410-A
Species-specific gyrB-PCR-RFLP differentiated all members of the
M. tuberculosis complex
63
M. bovis
RAPD-PCR
Four random primers and t w o
selected primers were used for
RAPD-PCR
Although RAPD-PCR identified 98 different patterns of 98
geographically dispersed isolates from Mexico, the method w a s
not able to differentiate M. bovis isolates by epidemiological
details or identify a common source of infection
82
M. bovis
Species-specific
multiplex PCR
Multiplex PCR of repeat-sequence of
M. tuberculosis hsp65f o l l o w e d by
solid-phase probe capture
hybridisation
Multiplex PCR of M. bovis hsp65 detected down to 4 CFU
equivalents in milk and nasal swabs of naturally and
experimentally infected cattle
bp
c
CFU
DNA
DR
ERIC
G
GTG
IS
LCx
LHS
5
5
base pairs
cytosine
colony-forming units
deoxyribonucleic acid
direct repeats
enterobacterial repetitive consensus sequences
guanine
guanine, thymidine, guanine
insertion sequence
ligase chain reaction
left-hand-side
PCR
PCR-SSCP
PFGE
PGRS
Pzase
RAPD-PCR
RE
REA
RFLP
RHS
RNA
5
129
: polymerase chain reaction
: polymerase chain reaction-single stranded conformational polymorphism
: pulse field gel electrophoresis
: polymorphic GC-rich repeat sequences
:pncAgene
: random amplified polymorphic DNA PCR
: restriction enzyme
: restriction enzyme analysis
: restriction fragment length polymorphism
: right-hand-side
: ribonucleic acid
documented in the comprehensive review of the use of
molecular epidemiology for tuberculosis by Van Soolingen
(138). Several methods of restriction enzyme analysis (REA),
RFLP, random amplified polymorphic DNA (RAPD) analysis,
PCR-ribotyping and multiplex PCR for several insertion
sequences (IS) and genes have been developed and evaluated
under experimental and field conditions, although most are
laborious, technically demanding and not yet standardised.
Fortunately, Cousins et al. have developed specific
recommendations towards a standardised protocol for DNA
316
fingerprinting of Mycobacterium
spp. that, in conjunction
with clinical field investigations, are expected to improve
research addressing the maintenance and transmission of
M. bovis in domestic and sylvatic animal populations (32). For
M. bovis isolates with three or more copies, IS6110-RHS RFLP
is adequate, even though IS6110-LHS RFLP has been shown
to be more sensitive. However, if the isolate has two or fewer
copies of IS6110, spoligotyping is recommended followed by
polymorphic GC-rich repeat sequence (PGRS)-RFLP analysis
for maximum sensitivity in discriminating strains (32), using
computer software for standardised computer-assisted
analysis and documentation of the banding patterns. Use of
standardised protocols will facilitate global comparisons and
tracking of the transmission dynamics of M. bovis.
Combinations of multiple RFLP analysis and spoligotyping
were found to yield the best differentiation among field strains
of M. bovis. Further refinements for molecular fingerprinting
of Mycobacterium
spp. are underway and include
genome-based
fluorescent
amplified-fragment
length
polymorphism (FAFLP) using two restriction endonucleases
(51) and analysis of whole genomic relatedness through
high-density DNA microarrays ( 7 ) . Both these procedures
offer an enhanced ability to differentiate among strains
without having to use technically demanding and laborious
procedures.
Conclusions
Modem technology and molecular biology have affected
virtually every form of discovery, confirmatory or
exclusionary diagnostic test, both in vivo and in vitro, often
significantly enhancing the quality of the assays or providing a
better understanding of host and/or M. bovis interactions.
Valid study designs to evaluate diagnostic tests in the
laboratory as well as under field conditions are essential for
accepting or rejecting completely new tuberculosis
diagnostics. Despite protein purification and molecular
cloning, no individual mycobacterial proteins or cocktail of
recombinant proteins have been found to eliminate the
specificity problems of PPD or to elicit DTH-like responses.
However, a M. tuberculosis 9 kDa protein, DPPD, was recently
found to detect a strong and specific DTH having no
cross-reactivity with other pathogenic or environmental
mycobacteria, thus providing the first putative specific and
sensitive alternative reagent for the diagnosis of tuberculosis.
Thus, modern molecular approaches to identify and clone
DTH diagnostic antigens may provide new reagents for
detecting M. bovis-infected livestock with equal or greater
sensitivity and specificity than current PPD antigens. This may
even allow vaccination of animals against tuberculosis while
discriminating M, bovis infected from vaccinated populations.
Assays based on PCR using formalin-fixed paraffin-embedded
sections with typical tuberculous lesions are highly specific for
M. tuberculosis complex mycobacteria and more rapid than
bacterial culture by three to four weeks. The use of
Rev. sci. tech. Off. int. Epiz., 20 (1)
immunohistochemical and PCR-based procedures offers
rapid, specific confirmatory tests for M. bovis infection,
especially on sections or tissues with typical tuberculous
lesions and acid-fast bacilli, or when herd history, tuberculin
test results and necropsy findings are available.
Most laboratories recommend the use of both an agar-based
medium (Middlebrook 7 H 1 1 ) and an egg-based medium
(Stonebrink or Löwenstein-Jensen with pyruvate) for primary
isolation. Modified Middlebrook 7H11 medium, B83
medium and radiometric detection have all improved the
recovery rate and speed of culture when used in conjunction
with standard specimen processing procedures. Identification
of isolates may be performed by standard procedures
including acid-fast staining, colony morphology and a panel
of biochemical tests such as susceptibility to 2-thiophene
carboxylic acid hydrazine (TCH) and isoniazid which require
an additional three to four weeks. However, the use of
monoclonal antibodies or genetic techniques now allow
definitive identification of M. bovis and DNA fingerprinting of
the isolate to be performed in one day.
Extensive evaluation of purified specific antigens as well as
crude M. bovis antigens for the serological detection of cattle
infected with M. bovis has revealed that the great majority of
serological assays are not as accurate as in vivo or in vitro
cell-mediated immunity tests. Serological tests for
tuberculosis should be used to complement diagnostics based
on cell-mediated immunity.
The comparative sandwich bovine IFNy ELISA for M. bovis
(using PPD-B and PPD-A) has probably had the most impact
on improving the specificity and sensitivity of simplified and
rapid in vitro cell-mediated immunity diagnostic tests (143).
This test has been evaluated world-wide and found to be
useful as a primary tuberculosis test under some conditions,
and as a complement to skin-testing in most countries.
Adaptations and refinements of the sandwich bovine IFNy
ELISA concept have steadily allowed investigators to explore
the spectrum of bovine host cell-mediated immune responses
of T cell subsets to a wide variety of crude and highly purified,
recombinant or synthetic antigens with specific epitopes. The
T cells appear to recognise a varying array of antigens, perhaps
low molecular weight secreted antigens, during the stages of
M. bovis infection of cattle, especially during early stages of
infection. In the future, these early-stage secreted antigens
could potentially be applied for diagnosis of active bovine
tuberculosis.
Methods of DNA fingerprinting have evolved rapidly and
deepened the understanding of naturally occurring
tuberculosis in domestic or sylvatic mammals. Several
methods of REA, RFLP, RAPD, PCR-ribotyping and multiplex
PCR for several insertion elements and genes have been
developed and evaluated under experimental and field
conditions. Fortunately, specific recommendations towards a
317
Rev. sci. tech. Off. int. Epa., 20 (1)
standardised
protocol
for
DNA
fingerprinting
of
Mycobacterium spp. have been made that are expected to
improve understanding of the epidemiology, maintenance
and transmission of M. bovis in conjunction with clinical field
investigations in domestic and sylvatic animal populations.
Acknowledgements
The author wishes to acknowledge the generous clerical
support of Ms. Jennifer Kristynik and technical support of
Ms. Roberta Pugh.
Significant progress has been made to improve the quality of
the in vivo and in vitro tuberculosis diagnostics for domestic
and sylvatic animals and additional progress is expected in the
near future.
Diagnostic in vivo et in vitro de l'infection due à
Mycobacterium
bovis
L.G.Adams *
Résumé
Le test intradermique d'hypersensibilité retardée utilisant un dérivé protéique
purifié issu de la mise e n culture de M. bovis ou de M. avium est l'épreuve la plus
utilisée pour le dépistage de la tuberculose ou le diagnostic de l'infection à
M. bovis c h e z les bovins. De n o m b r e u s e s améliorations ont été apportées au test
tuberculinique initial et les méthodes moléculaires visant à identifier et à cloner
les antigènes pourront accroître la spécificité et la sensibilité des tests
intradermiques d'hypersensibilité retardée. R é c e m m e n t , g r â c e a u x derniers
progrès technologiques, de nouvelles t e c h n i q u e s diagnostiques in vitro f o n d é e s
sur la détection d'anticorps, s u r l'immunité à médiation cellulaire ou s u r des
s o n d e s nucléiques ont été mises au point, assurant un diagnostic plus rapide que
la culture bactériologique. Le choix de la méthode diagnostique doit être effectué
en fonction de la population cible (animaux apparemment sains ou t r o u p e a u
présentant une forte p r é v a l e n c e de c a s cliniques) et de l'objet de la r e c h e r c h e
(sélection d'animaux sains ou confirmation de l'infection dans les cas s u s p e c t s ) .
D'autre part, les é p r e u v e s diagnostiques doivent être évaluées en faisant appel à
des groupes témoins s o i g n e u s e m e n t sélectionnés, dont la répartition entre
animaux infectés et non infectés soit représentative de la population cible.
Mots-clés
Bovins - Dérivé protéique purifié - Diagnostic - Mycobacterium bovis - Tuberculine Tuberculose.
Diagnóstico in vivo e in vitro de la infección debida a
Mycobacterium
bovis
L G . Adams
Resumen
La prueba intradérmica de hipersensibilidad retardada c o n uso de un derivado
proteico purificado a partir de un cultivo de M. bovis o M. avium es la t é c n i c a más
utilizada para diagnosticar la tuberculosis o detectar la infección por M. bovis en
el ganado v a c u n o . La prueba original de la tuberculina ha ¡do experimentando
318
Rev. sci. tech. Off. int. Epiz., 20 (
sucesivas mejoras, y el uso de t é c n i c a s moleculares para identificar y clonar
antígenos puede dotar a esta prueba dérmica de m a y o r especificidad y
sensibilidad. Los últimos a v a n c e s tecnológicos han propiciado la elaboración de
n u e v a s t é c n i c a s in vitro, como las de diagnóstico por anticuerpos, por la
inmunidad mediada por células o por reconocimiento de ácidos nucleicos,
métodos todos ellos que permiten diagnosticar la enfermedad con más prontitud
que un cultivo bacteriológico. Para elegir una t é c n i c a de diagnóstico conviene
tener m u y presente tanto la población que se analiza (animales en apariencia
sanos o rebaños con elevada prevalencia de casos clínicos, por ejemplo) como el
objetivo de los análisis (por ejemplo, la selección de animales sanos o la
confirmación de la infección e n c a s o s s o s p e c h o s o s ) . Por otra parte, a la hora de
evaluar una prueba de diagnóstico es n e c e s a r i o elegir c o n sumo cuidado una
población de control que sea representativa de la población problema (en cuanto
a la proporción relativa de ejemplares infectados y no infectados).
Palabras clave
Bovinos - Derivado proteico purificado
Tuberculina - Tuberculosis.
- Diagnóstico -
Mycobacterium bovis -
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