Advances in bovine tuberculosis diagnosis and pathogenesis: what policy makers need to know.

Abstract

The mainstay of tuberculosis diagnosis in cattle and deer has been the tuberculin skin test. Recent advances have allowed the incorporation of blood based assays to the diagnostic arsenal for both cattle and deer. Use of defined and specific antigens has allowed for improved specificity of cell mediated assays in both cattle and deer and advances in antibody tests for tuberculosis have potential for use in free-ranging and captive cervid populations. Combined use of blood-based assays with skin testing will require further understanding of the effect of skin testing on the accuracy of blood based assays. Models of experimental infection of cattle have allowed for increased understanding of natural disease pathogenesis. Differences likely exist; however, between cattle and deer in both disease distribution and primary route of inoculation in naturally infected animals.

Full text

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Wildlife Disease and Zoonotics
Michigan Bovine Tuberculosis Bibliography
and Database
University of Nebraska - LincolnYear 
Advances in Bovine Tuberculosis
Diagnosis and Pathogenesis: What
Policy Makers Need to Know
Mitchell V. Palmer∗
W. Ray Waters†
∗Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricul-
tural Research Service, USDA, 2300 Dayton Avenue, Ames, IA
†
This paper is posted at DigitalCommons@University of Nebraska - Lincoln.
http://digitalcommons.unl.edu/michbovinetb/69

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Advances in bovine tuberculosis diagnosis and
pathogenesis: What policy makers need to know
Mitchell V. Palmer*, W. Ray Waters
Bacterial Diseases of Livestock Research Unit, National Animal Disease Center,
Agricultural Research Service, USDA, 2300 Dayton Avenue, Ames, IA 50010, USA
Abstract
The mainstay of tuberculosis diagnosis in cattle and deer has been the tuberculin skin test. Recent advances have allowed the
incorporation of blood based assays to the diagnostic arsenal for both cattle and deer. Use of defined and specific antigens has
allowed for improved specificity of cell mediated assays in both cattle and deer and advances in antibody tests for tuberculosis
have potential for use in free-ranging and captive cervid populations. Combined use of blood-based assays with skin testing will
require further understanding of the effect of skin testing on the accuracy of blood based assays. Models of experimental
infection of cattle have allowed for increased understanding of natural disease pathogenesis. Differences likely exist; however,
between cattle and deer in both disease distribution and primary route of inoculation in naturally infected animals.
Published by Elsevier B.V.
Keywords: Cattle; Cervidae; Deer; Diagnosis; Mycobacteria; Mycobacterium bovis; Pathology; Tuberculosis
1. Issues related to diagnosis—advances since
the Mycobacterium bovis 2000 conference
A workshop of the M. bovis 2000 conference on
immunodiagnosis concluded that invitro IFN-g-based
tests and improved antigens should be evaluated for
use in TB eradication campaigns. At that time,
antibody-basedtestsforcattlewerelackingsensitivity,
yet development of improved platforms and antigens
was encouraged. It was acknowledged that antibody-
based tests are useful for diagnosis of tuberculous
deer.
Since the last conference, the IFN-g assay has been
widelyadaptedasafield testforbovineTBinmultiple
countries, either as a follow-up to or in parallel with
tuberculin skin testing. Large scale field trials of the
IFN-g assay in Australia in the early 1990’s indicate
that the sensitivity/specificity of the assay are 93.6/
96.3%, respectively (Wood et al., 1992). Additional
studies indicate that the sensitivity of the assay ranges
from81.8to100%andthespecificityfrom94to100%
(reviewed by Wood and Jones, 2001). In areas
where TB prevalence is low, concern exists that the
specificity of the IFN-g assay, using standard
PPD antigens, is insufficient for program needs.
www.elsevier.com/locate/vetmic
Veterinary Microbiology 112 (2006) 181–190
* Corresponding author. Tel.: +1 515 663 7474;
fax: +1 515 663 7458.
E-mail address: mpalmer@nadc.ars.usda.gov (M.V. Palmer).
0378-1135/$ – see front matter. Published by Elsevier B.V.
doi:10.1016/j.vetmic.2005.11.028
This article is a U.S. government work, and is not subject to copyright in the United States.

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For example, officials in Michigan, USA initiated
IFN-g testing as a follow-up to tuberculin skin testing
in 2004, with 2332 tests conducted during that year.
Test performance is monitored separately intwozones
within Michigan (i.e., locales based on TB preva-
lence). In 2004, test specificity was significantly
greater in the TB endemic zone (96.9%) compared to
the TB disease-free zone (?90.8%) (Larry Judge,
USDA, APHIS, personal communication). The unex-
pected lower specificity in the TB-free zone limits the
use of the test in such areas due to higher than desired
indemnity costs. To overcome this problem a
validation study has been implemented in Michigan
to evaluate the use of rESAT-6/CFP-10 for improving
test specificity. Possibly, use of specific antigens will
improve the specificity of the test and prove useful in
areas of low TB prevalence.
Application of antibody-based tests has been
extended to use with TB surveillance of free-ranging
white-tailed deer in Michigan. Two advances have
madethispossible.First,acaptureandreleasestrategy
in the TB endemic area was proven feasible. It was
determined that a reasonable percentage of free-
ranging deer could be captured, bled, and radio-
collared with minimal injuries. Secondly, antibody-
based tests performed satisfactorily for TB surveil-
lance of free-ranging white-tailed deer using blood
samples collected by hunters. Antibody-based tests
are particularly appealing for surveillance of captured
free-ranging populations as test formats amenable to
animal-side evaluation are now available. Addition-
ally, serum can be stored for later analysis without
need for immediate processing. This allows batch
processing, application of confirmatory tests, option
for allotment to multiple laboratories, and archiving
for future tests. Antibody-based tests are also being
evaluated for use in TB surveillance of free-ranging
Eurasian badgers (Meles meles) in the UK and elk
(Cervus elaphus nelsoni) in Canada. Similar applica-
tions may prove useful for other free-ranging
populations (e.g., cape buffalo, lions, and elephants).
Application of skin testing and slaughter surveil-
lanceprocedureshasgreatlyreduced TBprevalencein
countries with active eradication campaigns. Despite
successes, new diagnostic challenges resulting from
very low prevalence of disease and new wildlife
reservoirs have resulted in the need for development
and application of additional or improved tests. As
vaccination schemes are implemented, tests to distin-
guish vaccinated from infected animals will also be
required. The focus of this section on diagnosis is to
highlight advances over the past 5 years and identify
new applications for TB tests of cattle and wildlife.
2. Host response
The host response to the tubercle bacillus is
complex and broad, involving all aspects of the
immune system (Flynn and Chan, 2001; Kaufmann,
2001). The organism has evolved to avoid immune
clearance and induce chronic lesions ensuring
transmission by infectious aerosol droplets (North
and Jung, 2004). Paradoxically, lesions (i.e., granu-
lomas) are elicited as a mechanism to limit spread of
the bacillus, thereby preventing early demise of the
host. This complex host/pathogen interplay affords
both diagnostic challenges and opportunities.
Robust delayed type hypersensitivity (DTH) and
IFN-g responses are elicited upon experimental and
natural infection with M. bovis. The bias of the
immune response to M. bovis is a T helper type-1
response as evidenced by IFN-g, interleukin (IL)-12,
and tumor necrosis factor (TNF)-a production to
pathogen-associated antigen and exacerbation of
disease when these cytokine pathways are disrupted
(e.g., genetic deficiencies or inhibitory therapies)
(Flynn, 2004). Thus, a mainstay for TB diagnosis is
evaluation of these cell-mediated responses either by
skin test (Monaghan et al., 1994) or IFN-g-based
assays (Wood and Jones, 2001). Mycobacterial
infections also induce antibody responses. In general,
specific antibody responses are most easily detected
late in disease and correlatewith severe progression of
disease (Plackett et al., 1989). With improving
technologies,thecurrentdogmaofearlycell-mediated
and late humoral responses to mycobacterial infec-
tions may be challenged (Koets et al., 2001). Indeed,
intratonsilar inoculation of cattle with Mycobacterium
avium subsp. avium, M. avium subsp. paratubercu-
losis, Mycobacterium kansasii, or M. bovis induces an
antibody response detectable within 2–4 months of
inoculation;however,the intratonsilar routemay favor
early antigen/B cell interactions facilitating produc-
tion of specific antibody (Waters et al., 2003, 2004a;
Waters et al., Unpublished observations).
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3. Complex versus specific antigens
Over the past century, the predominant antigens
used for TB diagnosis have been complex, ill-defined
whole cell lysates and culture filtrates, particularly
tuberculin.Complexpreparationsprovideawidearray
of antigens for presentation to lymphocytes and are
representativeof the range of antigens presented to the
host upon infection. The most widely used antigen for
diagnosis of tuberculous cattle is purified protein
derivative (PPD) prepared from heat-killed cultures of
mycobacteria. PPD provides excellent sensitivity for
diagnostic tests yet lacks specificity due to epitopes
shared by various mycobacteria and other closely
related bacteria. Either in vivo (skin test) or in vitro
(IFN-g) comparisons made between responses to M.
bovis derived PPD and M. avium derived PPD are
beneficial for differentially diagnosing responses to
the respective mycobacteria. Improved specificity
may be obtained through use of defined antigens. Of
particular relevance for cattle are antigens that can be
used to distinguish between M. bovis and M. avium
infection. Other mycobacterial species commonly
isolated from cattle within the USA include:
Mycobacterium kansasii, Mycobacterium fortuitum,
Mycobacterium smegmatis, Mycobacterium pulveris,
Mycobacterium gadium, Mycobacterium terrae com-
plex, and Mycobacterium duvalii (Beth Harris,
personal communication). Responses generated by
exposure to non-tuberculous mycobacteria and other
related non-mycobacterial species might be of greater
significance for diagnostic tests used with cervids. A
prime exampleis reindeer (Rangifer tarandus) that are
notably prone to cross reactive responses that interfere
with TB test interpretation using standard PPD
antigens.
ESAT-6 and CFP-10 are potent IFN-g-inducing
antigens of tuberculous mycobacteria. These two
proteins are co-secreted and form a tight 1:1 complex
upon export (Renshaw et al., 2002). Genes for ESAT-6
and CFP-10 are absent in many environmental, non-
tuberculous mycobacteria as well as in the TB vaccine
strain, M. bovis BCG. Use of ESAT-6 and/or CFP-10
as antigens in IFN-g-based TB assays enhances
specificity when compared to use of M. bovis PPD
(Pollock and Andersen, 1997; Pollock et al., 2000;
Vordermeier et al., 2001; Buddle et al., 2001, 2003).
Unfortunately, genes for EAST-6 and CFP-10 are
present in some non-tuberculous mycobacteria, most
notably M. kansasii, M. marinum, M. leprae, and M.
smegmatis (Gey Van Pittius et al., 2001, 2002; Geluk
et al., 2002, 2004). Experimental infection of cattle
with M. bovis, M. avium subsp. avium, M. avium
subsp. paratuberculosis, or M. kansasii has demon-
strated that a fusion protein of ESAT-6 and CFP-10 is
recognized by M.bovisand M. kansasii-infected cattle
but not by cattle infected with either of the two M.
avium strains (Waters et al., 2004a; Waters et al.,
Unpublished observations). ESAT-6 has also been
used to discriminate between cattle naturally infected
with M. bovis and cattle sensitized/infected with
environmental, non-tuberculous strains of mycobac-
teria or vaccinated for paratuberculosis (Pollock and
Andersen, 1997; Buddle et al., 2003). In Ireland
studies have demonstrated the potential for ESAT-6 as
askintestantigen;however,largequantitiesofantigen
(up to 400 mg) and extended time to reading (i.e., 5
days instead of 3 days for standard antigens) are
required for best results (Pollock et al., 2003). It
should be noted that these potential pitfalls might be
overcome with use of adjuvants or additional specific
antigens.
A relatively large number of M. tuberculosis
complex-specific antigens
characterized in the late 1990s. Clearly, ESAT-6 and
CFP-10 are leading candidates for improved antigens
for use with cellular-based assays. Recently, Vorder-
meier et al. (2004) demonstrated that use of a fusion
protein of ESAT-6, CFP10, and detoxified Bordetella
pertussis adenylate cyclase improves the frequency of
responding cells in an IFN-g assay. Other antigens
such as Ag85, MPB83, MPB64, MPB70, Phos1
(38 kDa antigen), and somatic stress proteins have
been evaluated for use in cellular based assays with
variable success (Fifis et al., 1994; Vordermeier et al.,
1999; Rhodes et al., 2000; Pollock et al., 2001; Buddle
et al., 2003). Recent advances in whole genome
sequencing of M. bovis (Garnier et al., 2003) and
related mycobacteria (e.g., M. avium subsp. avium and
M. avium subsp. paratuberculosis) have resulted in
identification of numerous additional putative anti-
genic targets. Use of antigen cocktails and fusion
proteins may be employed to enhance the sensitivity
of specific antigen based tests. For instance, Lyash-
chenko et al. (2000) developed an antibody-based test
using complex and defined antigens. This test (multi-
werediscoveredand
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antigen print immunoassay or MAPIA) is useful for
screening responses by various host species to
determine antigen recognition patterns and kinetics
of responses to various antigens during the course of
infection and even in response to antibiotic therapy.
Optimal antigens as defined by MAPIA are then used
in a lateral flow immunoassay format or ‘‘Rapid Test’’
(Greenwaldetal.,2003).TheRapidTestiseasytouse,
amenable to animal side testing, adaptable for multi-
species use, and results are obtained within 15 min.
This strategy has proven promising for use in various
species including white-tailed deer, Eurasian badgers,
cattle, and various zoo species (Greenwald et al.,
2003; Lyashchenko et al., 2004; Waters et al., 2004b;
Lyashchenko, personal communication). As with
MAPIA and Rapid Test, future diagnostics including
cell- and antibody-based tests will likely employ the
useofcomplexantigensfortestsensitivityanddefined
antigens for test specificity.
4. Effects of skin test on cell- and antibody-
based assays
Injection of PPD for skin testing elicits a temporary
suppression of in vivo responsiveness to mycobacter-
ial antigens that may confound interpretation of
subsequent tests (Lepper et al., 1977; Radunz and
Lepper, 1985; Thom et al., 2004). Such suppression is
present from 4 to 60 days after injection (Radunz and
Lepper, 1985; Doherty et al., 1995). Mechanisms
underlying this reduced responsiveness are unclear;
however, both cellular and humoral factors have been
proposed. Practically, this phenomenon limits the
versatility of skin testing and is a forewarning of the
impact of antigen administration on subsequent in
vitro-based testing.
As with DTH responses, lymphocyte proliferative
responses are suppressed immediately following
injection of PPD in M. bovis-infected or sensitized
cattle (Doherty et al., 1995; Hall and Thoen, 1983). In
contrast, antibody responses are enhanced by PPD
injection, with peak effects 2 weeks after injection
(Lyashchenko et al., 2004). Effects of skin testing on
the M. bovis-specific IFN-g response, however,
remain unclear. Most studies indicate that IFN-g
responses are either not affected or slightly boosted
immediately after skin testing (i.e., 7–28 days later)
(Buddle etal.,1995;Dohertyetal., 1995; Rotheletal.,
1992; Gormley et al., 2004). Ryan et al. (2000)
determined that the IFN-g test is reliablewhen applied
8–28 days after skin testing; however, responses at
these time points were not compared to pre-skin test
responses. Rothel et al. (1992) found a marked decline
in the IFN-g response to M. bovis PPD at 7 days after
skin test; however, this decline was followed by an
enhanced response. Using M. bovis sensitized cattle,
Whipple et al. (2001) found that PPD specific IFN-g
responseswere boosted7daysafterskin test.Injection
of M. bovis PPD for the caudal fold test in 19
experimentally infected calves resulted in slightly
boosted IFN-g responses 3–5 days after injection and
responses from 7 to 55 days later that were similar to
pre-skin test values (Palmer et al., Unpublished
observations).
5. Issues related to pathogenesis
The pathogenesis of bovine tuberculosis is not as
well understood as the pathogenesis of tuberculosis in
humans. Advances in the field of human tuberculosis
have been made using various small animal models of
M. tuberculosis infection. Extrapolations of findings
from small animal models have been made to bovine
tuberculosis. Such extrapolations may not always be
appropriate as there are profound differences in host
susceptibility, host immune response, environment,
host ecology, and behavior. The current discussion
will deal primarily with studies in cattle and cervids.
6. Route of infection and lesion distribution
With some exceptions, it is agreed that cattle
become infected with M. bovis by either the oral or
respiratory routes (Neill et al., 1994). The oral route is
likely most important in calves nursing tuberculous
cows, while the respiratory route is most common in
cattle in general (Neill et al., 1994). In the late 1990’s,
surveys of tuberculous cattle in Great Britain (Phillips
et al., 2003) revealed that 67% of tuberculous lesions
were within the lungs and pulmonary lymph nodes
(tracheobronchial and mediastinal). Cranial lymph
node lesions were observed in 39%, while only 8% of
lesions involved mesenteric lymph nodes. Although
M.V. Palmer, W.R. Waters/Veterinary Microbiology 112 (2006) 181–190 184