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ORIGINAL RESEARCH
published: 01 October 2019
doi: 10.3389/fvets.2019.00328
Frontiers in Veterinary Science | www.frontiersin.org 1October 2019 | Volume 6 | Article 328
Edited by:
Dirk Werling,
Royal Veterinary College (RVC),
United Kingdom
Reviewed by:
Kieran G. Meade,
Teagasc, The Irish Agriculture and
Food Development Authority, Ireland
Amanda Jane Gibson,
Royal Veterinary College (RVC),
United Kingdom
Bryce Malcolm Buddle,
AgResearch, New Zealand
*Correspondence:
Anthony Duignan
anthony.duignan@agriculture.gov.ie
Specialty section:
This article was submitted to
Veterinary Infectious Diseases,
a section of the journal
Frontiers in Veterinary Science
Received: 19 June 2019
Accepted: 13 September 2019
Published: 01 October 2019
Citation:
Duignan A, Kenny K, Bakker D and
Good M (2019) Tuberculin PPD
Potency Assays in Naturally Infected
Tuberculous Cattle as a Quality
Control Measure in the Irish Bovine
Tuberculosis Eradication Programme.
Front. Vet. Sci. 6:328.
doi: 10.3389/fvets.2019.00328
Tuberculin PPD Potency Assays in
Naturally Infected Tuberculous Cattle
as a Quality Control Measure in the
Irish Bovine Tuberculosis Eradication
Programme
Anthony Duignan 1
*, Kevin Kenny 2, Douwe Bakker 3and Margaret Good 4
1Department of Agriculture, Food and the Marine, Dublin, Ireland, 2Central Veterinary Research Laboratory, Department of
Agriculture, Food and the Marine, Celbridge, Ireland, 3Independent Researcher and Private Consultant, Lelystad,
Netherlands, 4Independent Researcher and Private Consultant (Retired From Department of Agriculture, Food and the
Marine), Dún Laoghaire, Ireland
The Irish Bovine Tuberculosis (bTB) eradication programme operates under national
legislation and fulfills OIE and EU trade requirements. Tuberculin purified protein derivative
(PPD), a preparation obtained from the heat-treated products of growth and lysis
of Mycobacterium bovis or Mycobacterium avium (as appropriate), is critical to the
diagnosis of tuberculosis (TB). Standardization of Tuberculin PPD potency, the relative
activity in sensitized animals compared to a reference standard, is essential to underpin
the reliability of certification for international trade and to ensure that disease eradication
programmes are effective and efficient. A Bovine International Standard Tuberculin PPD
(BIS) was established by the WHO in 1986 and is used to determine comparative
potencies of Tuberculin PPDs. Ideally, Tuberculin PPD potency should be evaluated in the
species in which the tuberculin will be used but due to practical difficulties in performing
potency assays in cattle, for routine PPD production, they are usually assayed in guinea
pigs. Low potency tuberculin PPD is less efficient and thus inferior for bTB diagnosis.
Difficulties experienced in the Irish bTB eradication programme have included the supply
of sub-standard potency, and thus inferior, bovine (M. bovis) Tuberculin PPD in the late
1970s. The purpose of this paper is to outline the critical role of Tuberculin PPD assays
carried out on naturally infected tuberculous cattle, as required by the OIE and under EU
legislation in the quality control for the Irish Bovine Eradication Programme. Such assays
ensure that the Tuberculin PPD used meets the diagnostic sensitivity and specificity
requirements to underpin a successful national eradication programme.
Keywords: quality control, tuberculin, PPD, tuberculosis, bovine, potency, Ireland
Duignan et al. Tuberculin PPD Potency Assays
INTRODUCTION
Bovine tuberculosis (bTB) is an important infectious disease
of cattle that constitutes a “One Health” concern as a public
health risk due to its zoonotic potential (WHO) (1), and has
significant economic and trade implications for the European
Union (EU) and the World Organization for Animal Health
[Office International des Epizooties (OIE)] (2,3). Accuracy and
reliability of a diagnostic test are critical in disease control
and eradication strategies (2–5). Detection of the causative
Mycobacteria, all members of the Tuberculosis complex (MTBC),
during the early stages of disease is dependent on a measurement
of a cell-mediated immune response in vivo or in vitro, as
circulating antibodies remain undetectable until later in the
disease progression (4,6). The OIE (3) and the European
Commission (2) recognize the in vivo intradermal tuberculin
test (7) as the primary official test for the diagnosis of TB-
infected animals. Annex B of the EU trade Directive (2)
defines Tuberculin as “Tuberculin purified protein derivative
(Tuberculin PPD, bovine or avian) is a preparation obtained from
the heat-treated products of growth and lysis of Mycobacterium
bovis or Mycobacterium avium (as appropriate) capable of
revealing a delayed hypersensitivity in an animal sensitized to
microorganisms of the same species.” The intradermal tuberculin
test methodology for the diagnosis of bTB is applied in
accordance with OIE guidelines in many different countries using
differing applications (3). The single intradermal tuberculin test,
cervical (SIT), or caudal fold (CFT), and the single intradermal
comparative tuberculin test (SICTT) are widely used to detect
MTBC infected animals (7) for many national programmes and
for international assurance of freedom from bTB (3). Continuous
evaluation of all elements, i.e., inputs, performance, and
outputs, of the national disease control/eradication programme
is essential to maintain effectiveness and ensure that the highest
possible standards are attained and maintained (8,9).
Evolution of Tuberculins
Dr. Robert Koch demonstrated that M. tuberculosis was the
causative organism of human tuberculosis (TB) in 1882. While
attempting to develop a cure for TB he first produced what
became known as Koch‘s old tuberculin (KOT) in 1890 from a
crude extraction of heat killed cultures of M. tuberculosis (10–13).
By 1891, KOT was being used for the diagnosis of TB in cattle
and various tests applied although there were sensitivity (Se) and
specificity (Sp) issues associated with it (4,12). The first major
improvement both in tuberculin production and consequent test
Se and Sp was when synthetic medium was used for bacillary
growth (11). Seibert introduced precipitation of tuberculo-
protein in 1934 and so the term PPD was introduced (13).
Tuberculin PPD had less impurities and could be standardized
based on protein content. However, standardization using
protein content does not necessarily correlate with the biological
activity which must be routinely estimated against a reference
standard (14).
The 2018 publication of Good et al. (4) detailed that in 1939
Buxton claimed that the occurrence of non-specific response
could be overcome by the use of a synthetic culture medium and
precipitation in the production of tuberculin and that Buxton
and Glower attributed a precision of 87–97% to the tuberculin
test and recommended the use of synthetic medium tuberculin.
The first instructions on the performance of the SICTT issued in
1942 and detailed the conduct of the SICTT to compare the cell-
mediated immune responses to separate intra-dermal injections
of avian (M. avium) and initially mammalian (M. tuberculosis)
and later bovine (M. bovis) Tuberculin PPD in each animal,
to increase the specificity of the test in response to ongoing
concerns over the occurrence of non-specific response in animals
apparently not infected with TB (false positive) (4). In 1947,
Francis confirmed that the test interpretation for the SICTT and
optimal time of reading was “based on a very large number of
trials followed by postmortem examination” (4).
In 1948, Paterson described the AN5 strain of M. bovis that
grew as vigorously and with equivalent production capacity on
synthetic medium as did M. tuberculosis (12). By the 1950s,
bovine Tuberculin PPD, produced from M. bovis strain AN5, was
increasingly replacing mammalian Tuberculin PPD produced
from M. tuberculosis and was being widely used for eradication
of bTB. The main advantage of bovine Tuberculin PPD was
an increased Se and Sp in the diagnosis of TB in bovines over
mammalian Tuberculin PPD. Hence the change to bovine PPD
in the British and Irish bTB eradication programmes in the mid-
1970s (15). In 1959, Paterson described tuberculin as the most
important diagnostic agent in eradication schemes for bTB and it
remains so today (4,16).
Potency is a measure of a Tuberculin PPD’s activity in
animals sensitized with a specified organism when compared
to a reference standard Tuberculin PPD (17). Ritchie pointed
out that, for an effective test, it is vital to use a tuberculin
of potency greater than that to which the majority of infected
animals will respond (18). The use of a highly potent bovine
tuberculin increases the sensitivity of the test (19) and the balance
of evidence appears to favor the use of Tuberculin PPD of
sufficient potency to facilitate detection of the maximum possible
number of TB infected cattle for effective eradication of the
disease (3,16). Hence, the recommendations of the OIE are
that national bTB eradication campaigns use doses of Bovine
PPD of up to 50,000 IU/ml (20). Fears are often expressed
that the use of a highly potent Tuberculin PPD will reduce
the specificity of tuberculin tests and increase the false positive
rate. Experience in Ireland, however, where test Sp has been
demonstrated mathematically in an accepted non-disease-free
population, as at least 99.95% meaning that only a fraction of
1% of the positive reactors to the SICTT are false positive and
where the reliability (index of the diagnostic ability of a test)
of the SICTT was determined, both in 1992 and 2011, to be
in the region of 97%, would indicate that these fears are not
realized (20). Experimental studies carried in Britain involving
injection of tuberculous and non-tuberculous cattle with different
strengths of PPDs demonstrated that the stronger the tuberculins,
the better the differentiation between specific reactions (due to
M. bovis infection) and non-specific reactions. (21). In 1993,
Dr. Louis O’Reilly, the head of the TB Irish Central Veterinary
research Laboratory in a report (unpublished) on an evaluation
of the issue of potency and false positive results in the Irish bTB
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Duignan et al. Tuberculin PPD Potency Assays
eradication programme pointed out that when Dutch tuberculin
with a labeled potency of 40,000 IU/ml, which when assayed in
Irish tuberculous showed 40–50,000 IU/ml was used in Ireland
between May 1979 and April 1991 no problems with specificity
of the test were encountered. He also commented that it was
“very unlikely that the use of more potent bovine PPD will
result in more false positive reactors. In fact, the numbers of
false positive reactors should fall.” Use of highly potent bovine
Tuberculin PPD has evidently not been an issue for Ireland,
where, despite additional use of ancillary testing and more severe
test interpretation reactor numbers have generally been falling
since 2000 (22–24). Indeed the most recent tender for the supply
of Tuberculin PPD (dated 17/05/2019) for the Irish programme
specified that “Liquid Bovine PPD Tuberculin” potency “must
not be <50,000” IU/ml “in tuberculous cattle (to ensure potency
as assayed and used in the Eradication Programme over the last
10 years)” (25). Likewise, in GB, which uses the same Tuberculin
PPD combination in the SICTT as Ireland, Goodchild et al. (26)
states that in GB SICTT Sp at animal level is 99.87% (ultra-severe
interpretation), and that 91.1–93.7% of reactors in GB are truly
TB infected thus demonstrating that the SICTT, even using high
potency bovine PPD as demonstrated by cattle assay (Table 1)
retains a very high Sp with few false positive responders.
International Standards
To ensure uniformity in Tuberculin PPD production and
use throughout the world, the WHO established International
Biological Standards for potency of tuberculins. An international
standard (IS) for mammalian (human) Tuberculin PPD,
prepared using M. tuberculosis, with an assigned potency of
50,000 IU per mg, was established by the WHO in 1952 (17,28).
Similarly, an IS for avian PPD tuberculin, with an assigned
potency of 50,000 I.U. per mg, was established by WHO in
1954 (29). Investigations in 1995 found the quality of the avian
PPD tuberculin IS to be satisfactory both, in terms of potency
and specificity per weight and the same standard is still used
today (29). The Potency of a candidate Tuberculin PPD is then
determined by comparing the skin reactions (after intradermal
injection) to those elicited by the appropriate reference standard
Tuberculin PPD of known potency in animals sensitized with a
corresponding antigen. Thus, the potency of bovine Tuberculin
PPD is estimated using animals sensitized to M. bovis and
the potency of avian Tuberculin PPD is estimated in animals
sensitized to M. avium. Potency is expressed in international
units (IU) per ml; this allows comparison of tuberculins
throughout the world. An international unit is a measure of the
biological activity in a stated amount of the IS (17).
In 1964, the EEC adopted the Dutch National Bovine
Standard, prepared from cultures of M. bovis, strain AN5, as
the EEC standard for bovine Tuberculin PPD and assigned it
a potency of 50,000 units called Community Tuberculin Units
(CTU) (30,31). An EEC working group had shown that the
human Tuberculin PPD IS was not suitable for potency estimates
of bovine Tuberculin PPD due to the differing dose response
characteristics of both tuberculins (32).
In 1976, the WHO began an evaluation of candidate bovine
Tuberculin PPDs to select a new Bovine International Standard
Tuberculin PPD (BIS) (30). In 1986, a Dutch bovine Tuberculin
PPD, produced in 1979 from cultures of M. bovis, strain AN5, was
accepted as the BIS. International collaborative assays in cattle
and guinea pigs against the old Dutch bovine tuberculin standard
(1964) established that this new BIS had a potency of 32,500
CTU/ml and that CTU and IU for bovine Tuberculin PPD are
equivalent (31).
The 2018 OIE Manual of Diagnostic Tests and Vaccines for
Terrestrial Animals (20), quoting the 1968 WHO Technical
Report Series No. 384 (33) states “potency testing should be
performed in the animal species and under the conditions in
which the tuberculins will be used in practice.” It also references
the 1985 WHO Technical Report Series No. 745 (34) which
provides that calibration of laboratory (in-house) “reference
preparations shall be done by a number of tests” against the
appropriate IS in “the animal species in which the tuberculin
is to be used” and that the “control of potency of successive
batches can then be carried out by biological assays in guinea-
pigs, using the laboratory reference preparation.” The 2007
report of the WHO Expert Committee on the selection and
use of essential medicines (35) also required that all tuberculins
should comply with WHO Technical Report Series No. 745 (34).
The OIE Manual (20) goes on to say “that bovine tuberculins
should be assayed in naturally infected tuberculous cattle. As
this requirement is difficult to accomplish, routine potency
testing is conducted in guinea-pigs. However, periodic testing
in tuberculous cattle is necessary and standard preparations
always require calibration in cattle” (20). In addition, the
routine use of cattle for potency assay purpose can be both
impractical and expensive due to the lack of availability of
naturally infected cattle or the costs associated with laboratory
infection, thus guinea pigs are used as the alternative. Paterson
recommended that guinea-pigs be used for the control at
preparation/manufacture with occasional check assays in cattle
but that if the type of tuberculin is changed or if a change
in character is suspected that appeal must be to the assay in
cattle (12).
Due to the limited supply of the BIS, the EU and OIE
recommended that national and “in-house” standard Tuberculin
PPDs be calibrated against the BIS and then commonly used for
national and routine production potency assays. In 1994, an Irish
National Bovine Standard Tuberculin PPD (INBS) was produced
at CVL Lelystad and there are considerable stocks still available.
Calibration against the BIS, both in guinea pigs sensitized with
living M. bovis and naturally sensitized cattle, has shown that the
INBS has a potency of 33,700 IU/ml (26). In 2018, Frankena et al.
(36) suggested a new model to calibrate national and “in-house”
reference standards for maximum accurately using 30 naturally
TB-infected cattle (target species) and to prove the precision
and accuracy of the potency estimate using 54 guinea pigs in 6
individual potency assays. The variability in potency estimates
can be reduced by repeating the guinea pig assay 5 or more times
on each sample (36).
Manufacture and Composition of
Tuberculins
Tuberculin PPD has been described as a poorly defined, complex
mixture containing more than 100 individual components in
various stages of denaturation (37,38). Depending on where the
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Duignan et al. Tuberculin PPD Potency Assays
TABLE 1 | Assays of bovine tuberculin PPDs in naturally TB-infected cattle: 2010–2017, PPDs used in the Irish programme; 2006, 2010, and 2018, INBS and BIS; 2004,
2008, trials involving PPDs in tuberculin testing.
Batch no. Year
manufacture
Protein content
mg/ml
Guinea-
pig assay
IU/ml
Bovine assay IU/ml
Year 95% confidence limits Standard
Potency* Potency Lower Upper
INBS 1994 1.0 33,700 2018 30,568 19,749 46,981 BIS
BIS 1979 1.0 32,500 2013 25,821 13,792 46,561 INBS
INBS 1994 1.0 33,700 2006 46,079 31,722 68,238 BIS
170312 2017 1.0 30,350 2017 39,098 23,359 66,470 INBS
171502 2017 1.2 23,380 2017 76,927 46,043 140,170 INBS
162408 2016 0.95 24,850 2017 35,961 23,595 55,056 INBS
161102 2016 1.42 24,210 2016 50,082 30,243 86,391 INBS
153501 2015 1.25 40,950 2016 70,250 42,339 125,726 INBS
143806 2014 1.04 31,140 2015 59,058 36,584 100,423 INBS
143813 2014 1.02 30,540 2015 58,424 37,698 94,453 INBS
141310 2014 1.02 30,940 2015 52,697 34,020 84,478 INBS
141304 2014 1.01 30,640 2014 59,062 30,037 129,421 INBS
134511 2013 1.04 27,950 2014 36,822 18,300 75,367 INBS
102401 2010 1.11 26,580 2014 59,062 30,037 129,421 INBS
132203 2013 1.24 30,880 2013 52,601 35,380 80,416 INBS
132206 2013 1.08 26,900 2013 37,054 24,837 55,611 INBS
120103 2012 1.32 24,280 2012 64,795 37,264 122,280 INBS
120101 2012 1.15 21,130 2012 47,772 27,908 85,194 INBS
110404 2011 1.33 21,880 2011 50,811 30,532 88,291 INBS
110404 2011 1.33 21,880 2012 81,239 33,751 266,063 INBS
112006 2011 1.24 24,060 2012 59,999 35,112 109,705 INBS
104012 2010 1.16 23,500 2011 38,738 23,128 65,840 INBS
100308 2010 1.38 26,200 2011 57,979 34,854 102,114 INBS
102414 2010 1.11 26,220 2011 41,509 21,299 83,991 INBS
LPa2008 0.16 3,400 2008 11,920 4,950 23,540 INBS
NPa2008 1.23 26,380 2008 61,840 31,570 136,000 INBS
HPa2008 3.11 66,700 2008 125,540 62,570 323,820 INBS
JNb2004 na 10,250 2005 11,552 6,202 20,779 INBS
FMb2004 na 16,500 2005 25,900 14,866 44,784 INBS
KSb2004 na 9,250 2005 28,747 16,535 49,754 INBS
HTb2004 na 31,500 2005 33,868 19,532 58,734 INBS
GRb2004 na 27,750 2005 45,003 25,217 81,062 INBS
LPb2004 na 24,500 2005 45,003 25,217 81,062 INBS
*The Guinea Pig potency is determined against the Bovine International Standard (IS) (32,500 IU/ml).
aGood et al. (19).
bGood et al. (27).
na, not available.
Tuberculin PPD is to be used, or if for export to international
markets, its specification must meet the relevant international
standard requirements laid down by the WHO, OIE, and EU
legislative requirements (2,20,35,39). Tuberculins must be
sterile and free from abnormal toxicity. They must also be
non-antigenic, i.e., non-sensitizing when injected, so as not to
cause reactions at later injections in TB-free animals. Tests for
sterility, safety and sensitizing effect are set out in the OIE
Manual of Diagnostic Tests and Vaccines for Terrestrial Animals
2018 (20).
Potency
During production manufacturers are required to determine and
control the potency of tuberculin batches in guinea pigs against
a reference standard (34,35). However, guinea pigs and cattle
have different dose response relationships and further, there is
frequently only limited agreement between the guinea pig and
cattle potency assays (31,40,41) (Table 1). Potency estimate
accuracy and agreement between calculations done in guinea pig
and cattle can be improved but at the undesirable expense of
conducting repeated assays using more cattle and more guinea
Frontiers in Veterinary Science | www.frontiersin.org 4October 2019 | Volume 6 | Article 328
Duignan et al. Tuberculin PPD Potency Assays
pigs (5,28,36). The degree of variability in the guinea pig
bioassay have been the subject of comment previously (38,42).
In recognition of this problem, Directive 64/432/EEC (2015) (2)
requires the fiducial limits of error (P=0.95) to be not <50%
and not more than 200% of the estimated potency. The estimated
potency must not be <75% and not more than 133% of the stated
potency for avian tuberculin and not <66% and not more than
150%, of the stated potency for bovine tuberculin and to comply
with Directive 2001/82/EC (2,43). The tuberculo-protein content
of the M. bovis Tuberculin PPD is adjusted based on guinea
pig potency assay to achieve the target potency not <20,000 IU
per ml in each final product batch. OIE recommends for bovine
Tuberculin PPD that “In cattle with diminished allergic sensitivity,
a higher dose of bovine tuberculin is needed, and in national
eradication campaigns, doses of up to 5,000 IU (i.e., 50,000 IU
per ml) are recommended” and thus bovine Tuberculin PPD with
target potency exceeding 20,000 IU per ml may be sought and
produced by manufacturers (20).
The method of sensitization of guinea pigs to M. bovis antigens
can influence the results of potency assays. Repeatedly, studies
have shown that the closest correlation with cattle assays is
achieved by sensitization of guinea pigs with living M. bovis i.e.,
in effect infecting these guinea pigs with M. bovis (32,44,45).
Sensitization of guinea pigs with heat-killed M. bovis or with live
M. bovis BCG gives less reliable results, presumably because the
full complement of antigens excreted during the mycobacterial
multiplication stages of active infection are not produced.
Likewise, potency assays performed in cattle sensitized with
heat-killed M. bovis are not reliable (46). Cattle experimentally
infected with living M. bovis are suitable for potency assays as
are naturally infected cattle. The advantage of using naturally
infected cattle from field bTB breakdowns is that this represents a
more complete spectrum of exposure and stages of infection that
will occur in naturally acquired infection than using a group of
homogenously infected cattle.
The specificity of each production batch of bovine Tuberculin
PPD is estimated in guinea pigs sensitized with heat inactivated
M. avium according to Fishers’ method (46). The skin responses
elicited by the bovine PPDs are compared to those of the
IS for avian PPD. The specificity of avian Tuberculin PPD is
estimated in guinea pigs sensitized with living M. bovis using the
same methodology.
Paterson, Haagsma et al., WHO, and OIE, recommended as
good practice to periodically check the results of the guinea
pig potency assays by estimating the potency of a proportion
of production batches in naturally or artificial infected cattle
(12,20,29–33). However, whilst this may be good practice and
was provided for in the original Directive 64/432/EEC (1964) (47)
and, in 1979, noted as essential by the experts in the EC sub-group
of the Scientific Veterinary Commission on tuberculins (32), it
was omitted when Directive 64/432/EEC was modified in 2002
(Commission Regulation 2002)1. The OIE Manual of Diagnostic
Tests and Vaccines for Terrestrial Animals (Chapter 2.4.6—
Bovine Tuberculosis) (20), still includes the recommendation to
1Commission Regulation (EC) No 1226/2002 of 8 July 2002 amending Annex B to
Council Directive 64/432/EEC OJ L 179, 9.7.2002, p. 13–18.
perform potency assays in tuberculous cattle, while reference is
made to WHO Technical Report Series No. 745 (34).
Notwithstanding attempts by the WHO, OIE and the EU to
standardize Tuberculin PPD production, quality and potency
estimation, qualitative and quantitative differences between
Tuberculin PPDs from different manufacturers exist. These
differences occur due to various factors such as differences
in manufacturing facility location, possible differences in
growth media and production seed-stock strain. Differences
in manufacturer’s potency calibration methods, including staff
experience and attention to detail, and, the quality of the
reference standard used, can affect assessed potency in guinea
pigs and consequential potency in cattle. In addition, the means
of sensitization of guinea pigs and the number of guinea pigs
used for assay by the manufacturer will affect the accuracy of the
potency estimate and further result in inter-laboratory differences
in potency estimates. These differences result in a wide variance
both in protein content and antigenic profile and thus, differences
in potency and specificity between various Tuberculin PPD
products are to be expected and these, plus the relative potency
of the avian and bovine Tuberculin PPDs used for SICTT and the
Interferon-γ(IFN-γ) assay, will have an impact on test efficacy,
Se, Sp, and Predictive Value (44,48–52).
The Irish bTB Eradication Programme
In Ireland, bTB is caused predominantly by infection with M.
bovis. The Department of Agriculture, Food and the Marine
(DAFM) manages the Irish bTB eradication programme which
includes annual screening of all cattle herds, prompt removal
of test positive animals (reactors) and animals removed for
epidemiological reasons by a Veterinary Inspector or animals
removed following the results of ancillary blood test(s) such as
the interferon gamma (IFN-γ) assay, post-mortem surveillance
by veterinary practitioners of all bovine carcases at slaughter
for human consumption, movement restrictions and further
consequential testing of infected herds (22–24). Good et al. in
2011 state that some 7% of cattle were positive to the single
intradermal test but not to the SICTT and that various pathogenic
mycobacteria e.g., Mycobacterium paratuberculosis subsp. avium,
and non-pathogenic environmental Mycobacteria such as M.
hiberniae, are abundant in the Irish environment, and cause
non-specific sensitization to bovine Tuberculin PPD (20,53).
Accordingly, the SICTT is the primary screening test employed
in the programme and entails ∼8.5 million animal tests each
year (7,22–24). Intradermal injections of 0.1 ml of avian (25,000
IU/ml) and bovine (30,000 IU/ml) Tuberculin PPD, as assessed in
guinea pigs (supplied by Prionics, Lelystad B.V.) are administered
in the mid-third of the neck; the skin thickness at the site
of the test is recorded at the time of injection and at test
reading 72 h [±4 h] later. The nature of any reaction and the
relative increase (measured in millimeters) in skin fold thickness
at each injection site is evaluated at test reading. Any animal
that displays clinical signs at the bovine injection site, such as
oedema, exudative necrosis, heat and/or pain, in response to
the injection of bovine tuberculin, at test reading is deemed test
positive and therefore a “reactor” regardless of relative increase in
accordance with the Directive 64/432/EEC (2015) (2). During the
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Duignan et al. Tuberculin PPD Potency Assays
late 1970s, Ireland experienced problems with the low potency
of tuberculin supplied for the programme. Subsequently, Ireland
changed Tuberculin PPD supplier in 1980 and developed strict
criteria for its requirements and incorporated potency assays on
naturally infected cattle as a quality control measure (23,24).
Tuberculin PPD Requirements for Irish bTB
Eradication Programme
Under Irish legislation, the only tuberculin that may be used
in the Irish bTB eradication programme, is that supplied by
DAFM. It must have marketing authorization (MA) from the
Health Protection Regulatory Authority (HPRA) in Ireland
in compliance with EU legislation (39,54). Prionics Lelystad
BV (previously Lelystad Biologicals BV or ID-Lelystad BV), in
Lelystad, The Netherlands, has supplied the avian and bovine
Tuberculin PPD used in the Irish programme since 1980
under tender.
Specification
The preparation, potency and labeling of Tuberculin PPD must
conform to Article 51 of Directive 2001/82/EC and as specified
in Directive 91/412/EEC (54). Storage must be at 4◦C but,
in accordance with the marketing authorization (MA), must
be stable at ambient temperatures for 14 days between +2◦C
and +37◦C.
Potency
Under the MA the total protein concentration of the Avian
Tuberculin PPD 2500 must be between 0.5 and 0.8 mg/ml and
that of the Bovine Tuberculin PPD 3000 between 1.0 and 1.4
mg/ml while at the same time:
- Bovine Tuberculin PPD supplied under MA should have a
potency of 3,000 IU/dose for a 0.1 ml dose [66–150% i.e.,
between 1,980 IU and 4,500 IU] when tested in guinea
pigs sensitized by living M. bovis, strain AN5 and, for the
most recent tender for the supply of tuberculin PPD (dated
17/05/2019), “not be less than 50,000” IU/ml “in tuberculous
cattle” and that “Potency will be determined by or on behalf
of the Contracting Authority representative and prior to
acceptance of any batch of tuberculin under the contract”
(25) and
- Avian Tuberculin PPD should have a potency of 2,500 IU/dose
for a 0.1 ml dose [75–133% i.e., between 1,875 IU and 3,325
IU] per dose when assayed in guinea pigs sensitized with heat
inactivated M. avium and
- the pairs of Tuberculin PPDs, for the SICTT, must not exceed
a maximum potency difference of 500 IU per dose (using the
potency as assessed in guinea pigs) between both (Avian and
Bovine Tuberculin) in the Tuberculin PPD Kit.
This stated Bovine PPD potency requirement exceeds the
minimum of 20,000 IU/ml specified in Directive 64/432/EEC
(2). The assays are carried out in guinea pigs as set out in
EU Directive 64/432/EEC (2) using “a reference preparation of
tuberculin (bovine or avian, as appropriate)” PPD “calibrated in
International Units” by or on behalf of Prionics Lelystad BV.
CATTLE POTENCY ASSAY
The potency of Bovine Tuberculin PPD 3000 supplied for use
in Ireland is estimated in Lelystad in guinea pigs sensitized
with living M. bovis relative to the BIS PPD on behalf of the
manufacturer. The potency of one or more supplied batches of
Tuberculin PPD is checked in Ireland in naturally infected cattle
each year against the INBS, which was calibrated in 1994 in cattle
and guinea pigs (28). The potency of Tuberculin PPDs used in
various trials involving tuberculin testing in Ireland has also been
assayed (19,27). The INBS tuberculin is also periodically assayed
against the BIS in naturally infected Irish cattle in Ireland and
in guinea pigs by Prionics Lelystad BV in accordance with EU
Directive 64/432 (2). A recent assay included the INBS against the
BIS in 2018 in naturally infected Irish cattle indicated a potency
of 32,265, an earlier assay in 2006 had shown a potency of 46,079
and an assay of the BIS against the INBS in 2013 had shown a
potency of 25,821 (Table 1). The results of these latter two assays
caused some concern. Subsequently, at the M. bovis conference
in Cardiff in 2014 (55) problems were reported where evident
visual differences and even non-visual deterioration of the BIS in
some ampoules supplied by the NIBSC was resulting in highly
variable potency assay results being obtained in guinea pigs,
when injecting identical amounts from different vials. Dr. Bakker
reported that in the deteriorating vials the BIS was no longer
completely water soluble and contained varying amounts of large
particles which affected the potency assay and while these could
be centrifuged (3,000×g) and removed there was then a loss
of antigen (55). Consequently, the NIBSC removed the visibly
deteriorated ampoules from their stocks and, the OIE established
in 2015 an ad hoc working group with the task of finding, a
new source of a bovine PPD and work is currently underway to
develop a new BIS.
In Ireland cattle for assays are chosen in accordance with the
Standard Operating Procedures for Tuberculin Potency Assays
on TB cattle in the isolation unit at the DAFM Research Farm
the purpose of which are to ensure the welfare of the cattle and
the integrity of the assays carried out. Cattle assays are subject to
strict individual project licensing conditions which are issued and
audited by the HPRA.
Cattle, from TB-infected farms of origin, which have given
a positive result to a SICTT, which have a skin-fold thickness
measurement increase at the bovine injection site, which is more
than 4 mm greater than the increase at the avian injection site
and are positive to IFN -γBOVIGAM R
(Prionics, Lelystad B.V.)
assay (56), are selected for assays at the isolation unit. For ease
of handling and husbandry, young steers from ∼6-months to 2-
years of age are usually selected. The interval between the SICTT
on the farm of origin and the potency assay must be at least 60
days (57). The animals are normally kept for a maximum of two
assays or for up to a year and replaced as necessary by further
field test positives as above.
Potency assays on batches of routine issue bovine Tuberculin
PPD are carried out under license 2–3 times each year depending
on availability of sufficient numbers of suitable field reactor cattle.
At each assay, the potencies of three selected test batches are
estimated against the IBNS bovine Tuberculin PPD with an
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Duignan et al. Tuberculin PPD Potency Assays
assayed potency of 33,700 IU/ml. Each ampoule of the freeze-
dried IBNS contains 1.8 mg PPD in a glucose phosphate buffer
containing phenol (28). Dilution to 1 mg/ml is prepared, by
adding 1.8 ml of distilled water. Isotonic phosphate-buffered
saline, pH 7.3 is used to prepare the 20% dilutions of all the
Tuberculin PPDs for the assay representing 0.2 mg/ml protein
concentration (30). Each of the 3 Tuberculin PPDs for assay
and the comparator INBS and/or BIS, as relevant, is used at
two dilutions corresponding to protein concentrations of 1.0 and
0.2 mg/ml.
Thus, there are eight tuberculin preparations (4 undiluted and
4 diluted) which are inoculated into each animal at four sites on
each side of the neck as shown in Figure 1. The distance between
the injection sites is ∼10–12 cm.
A group of eight cattle is required to rotate all 8 injections (i.e.,
the reference standard and 3 test PPDs each at two dilutions)
sequentially through each of the eight possible neck sites using
the Latin square design. This is necessary to take account of
the different sensitivity at different sites on the bovine neck
(12,17). The Tuberculin PPDs are allocated to sites based on a
randomized schedule laid out in advance in the assay worksheets.
For routine assays, three groups of eight cattle are used to increase
the reliability of the assay to acceptable levels of accuracy i.e.,
for the same reasons as recommended by Frankena et al. for the
guinea pig assay (36). A separate worksheet, each with a unique
pattern of allocation of Tuberculin PPDs to neck sites is used for
each group.
The injection sites are clipped and the skin-fold thickness
at each injection site is measured using a caliper with 1 mm
graduations at 0 h. Using McLintock syringes, 0.1 ml of each
Tuberculin PPD are injected intradermally. All aspects of each
test (tuberculin administration, initial and subsequent skin
measurement) on each assay animal are conducted by the same
veterinarian. Each injection site skin thickness measurement is
taken and recorded at 0 and 72 h. Table 2 shows an example of
the skin measurement data recorded for one of the 3 groups of 8
cattle on a recent assay.
FIGURE 1 | Injection sites with reactions, showing injection/measurement
sequence,1–4 (on each side of the neck), for bovine tuberculin PPD assay.
Measurements, recording increases at the various injection
sites for each dilution of PPD being assayed and the reference
standard, are analyzed using standard statistical methods for
parallel-line assays (58), using the GLM procedure in SAS v9.1
(59). Site of injection and side of the neck are included in the final
model if significant (P<0.05). The 95% confidence limits for
the relative potency are calculated according to Fishers’ method
(46). This analysis estimates the potency of the three routine issue
Tuberculin PPDs as compared to the IBNS Tuberculin PPD (46).
Potency is expressed in IU/ml, based on the potency of the INBS
at 1.0 mg/ml of 33,700 IU/ml as calibrated previously against the
BIS (28). Table 3 shows the results of the analysis for the assay
referred to on Table 2.
Guinea pigs and cattle have different dose response
relationships and unless multiple groups of cattle and guinea pigs
are used both accuracy of the potency estimate and the potency
correlation between the species is poor (11,28,29,32,36,54,60).
Notwithstanding multiple assays in each species, the potency
estimated in the guinea pig (five assays each with 9 guinea pigs),
differ from those obtained in cattle assays (three assays each
with 8 cattle) as evidenced by the results shown in Table 1. At
least some of the difference is likely to be due to the BIS being
used as the reference standard for the guinea pig assay. The
BIS, as stated previously has been deteriorating from prior to
2005 when it was first publicly reported (36), and has since
been giving highly variable potency assay results in guinea pigs,
when injecting identical amounts from different vials (55).
However, successive assays have shown (Table 1) that batches
that meet the required potency of 30,000 [66–150%] IU/ml in
guinea pig assays also attained or exceeded this potency when
assayed in cattle and indeed in recent years frequently attains or
exceeds the potency recommended by the OIE for use in bTB
Eradication programmes. From the results presented in Table 1,
it would appear likely that the assay results in guinea pigs are
underestimating the potency of the assayed PPDs by varying
amounts most likely depending on the degree of deterioration of
the BIS in the vial used for the assay.
DISCUSSION
As early as 1908, it was lamented that “some of the tuberculin
on the market is impotent and worthless” and Buxton also
commented on tuberculin quality in 1934 (60–63). In 2011, Good
(19) compared “the impact of different potencies of a single
bovine PPD tuberculin on the field performance of the” SICTT
and SIT and found “a significant difference in the number of
reactors detected using the high and low potency tuberculins.”
This study also found that the low potency tuberculin, although
not the lowest detected commercially available, missed detecting
animals which, having negative responses at the bovine injection
site, would individually have qualified for OIE certification as
bTB-free for export purposes, despite subsequently being found
to have multiple tuberculous lesions visible at routine slaughter
(3,5,19).
When the United Kingdom (UK) switched from using
Weybridge Tuberculin PPD to Dutch Tuberculin PPD, it was
found that the tuberculin manufacturing source influenced both
the Se and Sp of the SICTT (63). Data from international
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Duignan et al. Tuberculin PPD Potency Assays
TABLE 2 | Example of injection site measurement data for a group of 8 bovines in a potency assay on bovine tuberculin PPDs.
Group 2 Irish standard Test PPD L Test PPD M Test PPD N
1.0 mg 0.2 mg 1.0 mg 0.2 mg 1.0 mg 0.2 mg 1.0 mg 0.2 mg
Animal ID Hour *Inc Hour *Inc Hour *Inc Hour *Inc Hour *Inc Hour *Inc Hour *Inc Hour *Inc
0 72 0 72 0 72 0 72 0 72 0 72 0 72 0 72
1038 12.5 19 6.5 13 18 5 13.5 18 45 11 17 6 10 16.5 6.5 12 24 12 11 18 7 13 16 3
2190 11 16 5 9 12 3 10.5 14 3.5 9 12.5 3.5 10 15 5 10 14 4 9 12 3 11 16 5
1629 7 13 6 10 15 5 8 14 6 10 14 4 10.5 16 5.5 8 14 6 10 14.5 4.5 6 10 4
1664 8 16 8 8.5 16.5 8 7 13 6 7.5 15.5 8 9 19 10 7 13 6 9 19 10 8 15 7
1636 8 12.5 4.5 6 11 5 8 12 4 7.5 12 4.5 7 14 7 8 10.5 2.5 6.5 12 5.5 9 11 2
1658 9 16 7 9 15.5 6.5 8 16 8 11 18.5 7.5 9 20 11 8 13 5 12 19 7 9 13 4
2256 9 18 9 11 18.5 7.5 10 20 10 10 16 6 9 19 10 11 17 6 10 17.5 7.5 10 14.5 4.5
2175 7 12.5 5.5 9 12 3 9 16 7 9 15 6 8 14 6 9 13.5 4.5 9 18 9 7.5 12 4.5
*Increase in skin fold measurement millimeters at 72 h (72 h.mms−0 h.mms =Inc.mms).
TABLE 3 | Example of relative potency and calculated international units of the 3
test bovine tuberculin PPDs in Table 2.
Relative potency (%) International units (IU/ml)
95% confidence
limits
95% confidence
limits
Test PPD Mean Lower Upper Mean Lower Upper
L 109.3 543 223.6 36,822 18,300 75,367
M 175.3 89.1 384 59,062 30,037 129,421
N 118.2 59.1 244.2 39,839 19,921 82,288
studies indicate a sensitivity range, at individual animal level,
of 68–96.8% and 96–98.8% specificity for the CFT (80–91%
sensitivity and 75.5–96.8% specificity), for SIT and, for the
SICTT (55.1–93.5% sensitivity and 88.8–100% specificity) (7,
60,64). The caudal fold has been repeatedly determined to
be the least sensitive site available for intradermal test and
hence, the CFT requires higher potency tuberculin to achieve an
acceptable Se for use in bTB control/eradication programmes;
the mid third of the neck proved to be the most sensitive
site for the intradermal test (4). Differences in test sensitivity
and specificity are largely due to bTB prevalence, variation in
testing techniques, differences in tuberculin doses, Tuberculin
PPDs with differing antigenic profiles, Tuberculin PPD potency,
relative potency of avian, and bovine PPDs in the comparative
test, the interpretation of skin reactions, and the prevalence
of non-specific or cross-reactive antigens in the environment
(19,23,38,50). Different findings of various studies are attributed
to multiple factors including differences in study design, the
selection of animals in various stages of infection, frequency
of testing and most significantly, how the infection status of
the animals was determined. The specificity of the test will be
affected by sensitization to environmental mycobacteria or other
organisms that have shared antigens with M. bovis (65). The level
of cross-sensitization will vary from region to region. In Ireland,
exposure of cattle to multiple environmental mycobacteria may
result in cross-reactions to bovine PPD (53), nevertheless, as
previously stated, the reliability of the SICTT, being a relatively
crude index of the diagnostic ability of a test based on the Se and
Sp of the test in the environment in which it is used, has been
assessed as being in the region of 97% (20,66).
Few, if any, studies discussing the sensitivity range, and
comparing ante-mortem test outcomes in various countries or
regions or over time consider differences in the manufacturer
or potency of the tuberculin as critical to test Se and much of
the more recent literature in particular, seems to assume that all
tuberculins will perform equally whether in skin test or IFN-γ
assay (48,67–71). For example, the 2012 EFSA scientific opinion
states that the selection of the cattle populations, the bovine TB
testing history of the cattle and the prevalence of environmental
mycobacteria, may have influenced performance estimates in the
surveillance population samples used in their latent class analysis,
specifically mentioning the low sensitivity of the skin test in one
dataset (68). However, they fail to mention as pertinent that the
tests they compared in the datasets used tuberculin, avian and
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Duignan et al. Tuberculin PPD Potency Assays
bovine PPDs, from 3 different manufacturers and with, at least
the bovine Tuberculin PPD, at 2 different stated potencies, with,
at the time the data was generated, a different manufacturer’s
Tuberculin PPDs used in each jurisdiction. In the context of
tuberculin used in the IFN-γassay for example, Tameni et al. (51)
commented that wide fluctuations of the results of the IFN-γ
assay had been traced back to the use of different PPD batches
and that tuberculins were not prone to easy standardization
of their antigenic content. Casal et al. (69) compared the
performance of the SIT, ID-Vet IFN γ, and the Bovigam R
noting that “Over the 113 cattle with confirmed bTB (group
2), 32 (28.3%) were classified as positive reactors by Bovigam R
but negative to the SIT test;” and in the same group “36 cattle
(31.9%) were positive with Bovigam R
(0.05 cut-off point) but
negative to IDvet IFN-γassay (35% s/p cut-off point).” However,
similar results were achieved between the IFN-γassays applying
the 0.1 cut-off point in the Bovigam and the S/P ratio of 16 in the
IDvet test. These results were comparable to the results obtained
by de la Cruz et al. (70) who also found the IDvet IFN-γassay
less sensitive than the Bovigam R
but that the Se of the IDvet
IFN-γassay might be improved by adjusting the cut off points.
The Bovigam TB kit flier states that BOVIGAM Tuberculin
PPD produced by Prionics Lelystad B.V. uses bovine Tuberculin
PPD at a potency of 30,000 IU/mL and avian Tuberculin PPD
at a potency of 25,000 IU/mL (https://assets.thermofisher.
com/TFS-Assets/LSG/Flyers/animalhealth_flier_bovigam_tb_
CO121138.pdf). The IDvet brochure states that their IDvet
test for detecting the cellular response to Mycobacterium bovis
uses bovine PPD as the specific antigen source and avian PPD
as the non-specific antigens source with matched potencies
of bovine and avian tuberculins but the potency/ml is not
stated (https://www.id-vet.com/wp-content/uploads/2014/07/
brochure_IFNG_BovineTB_doc250.pdf). When discussing the
Se, Sp and efficacy for the detection of TB infected animals of
the various tests, neither set of authors considered the source of
the Tuberculin PPDs, avian, and bovine, the individual potency
or the relative potencies of these PPDs, in the possible reasons
for the differences in observed results of the test performances.
Similarly, Keck et al. (71) observed very low SIT positive rates
during two screening campaigns where the use of the Bovigam R
assay was found to increase the sensitivity of TB detection
by more than 30% over and above the SIT using the official
bovine Tuberculin PPD and the effect of the different PPDs
used was not discussed as a possible factor in the different Se
observed (71).
Largely, due to the ill-defined nature of the antigens in
Tuberculin PPD as well as the complexity of Tuberculin PPD
production, to date, there has been little progress in improving
Tuberculin PPDs to enhance test specificity and sensitivity (37,
52). Successful eradication of bTB has been achieved in many
countries by the rigorous application of tuberculin testing and
the culling of reactor cattle. While the quality of the Tuberculin
PPD used is undoubtedly critical for test efficacy for bTB control
and eradication programmes and to underpin certification of
disease freedom at animal and herd level, comparisons of
commercially available tuberculins, has shown the potency of
bovine tuberculins and, to a lesser extent, avian tuberculins
varied widely such that the majority would not have met the
required minimum dose of 2,000 IU if applied as the standard
0.1 ml dose (5,42). The use of tuberculins with inferior potency
has direct implications for the diagnosis of bTB and for the
surety of consequent certification of herd and animal disease
freedom (5,19). While the European Pharmacopeia, WHO,
OIE, and EU have established the standard for tuberculins
(2,20,33–35,39,43,54), there is no independent body
evaluating commercially available preparations or establishing
and maintaining standards of Tuberculin PPD potency akin
to The International Organization for Standardization (https://
www.iso.org) ISO which is designated to independently assess
and attest the standards claimed by the manufacturers. There
may be a potential role for the European and/or OIE Tuberculosis
Reference Laboratories in the verification of tuberculin potency.
It would reasonably be expected that when standards are not
complied with that the authorities should take steps to ensure
that such products are precluded from use. It would also
undoubtedly be desirable to have an alternative methodology
for PPD potency assay less dependent on infecting Guinea
Pigs and the availability of TB infected cattle. Due to potency
issues with tuberculin supply in the past and considering
the above publications demonstrating that potency is critically
important in test efficacy, Ireland has, using naturally infected
tuberculous cattle, maintained an independent check of the
potency of the bovine Tuberculin PPD supplied under the
Irish programme.
DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the
manuscript/supplementary files.
ETHICS STATEMENT
The Tuberculin PPD assays and use of animals therein was
reviewed by Department of Agriculture and Marine/University
College Dublin ethics committee as part of the Health Products
Regulatory Authority (HPRA) project authorization application
process. The HPRA is the competent authority in Ireland
responsible for the implementation of EU legislation (Directive
2010/63/EU) for the protection of animals used for scientific
purposes. HPRA are committed to ensuring that the care
and use of animals for scientific purposes is in line with
the 3R principles—Replacement, Reduction, and Refinement.
Accordingly, projects and individuals require authorization to
carry out research using animals including authorization of
the establishment where the animals are kept. The use of
animals for Tuberculin PPD potency assay, the persons who
perform the assays and the premises where the animals are kept,
are authorized (licensed) by the HPRA and subject to strict
individual project licensing conditions which include reporting
and annual audit.
AUTHOR CONTRIBUTIONS
AD conceived the study. AD, DB, and MG analyzed the data.
AD and MG carried out a literature search and wrote the initial
Frontiers in Veterinary Science | www.frontiersin.org 9October 2019 | Volume 6 | Article 328
Duignan et al. Tuberculin PPD Potency Assays
manuscript. All the authors participated in reviewing, editing,
read and approved the final draft, and collaborated in producing
the final version.
FUNDING
This work was funded entirely by the Department of
Agriculture, Food and the Marine as part of the bovine TB
Eradication Programme.
ACKNOWLEDGMENTS
The authors wish to thank staff at DAFM for sourcing infected
cattle for tuberculin assays, staff at DAFMs Research Facility and
particularly Eamon Costello (retired) and Colm Brady of the
DAFM for their assistance. The assistance of staff at of Prionics
Lelystad BV with the technical aspects of tuberculin production
and guinea pig assays is also much appreciated. The authors
also wish to thank Daniel M. Collins for his assistance with the
preparation of the manuscript and the figure.
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
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