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Indian Journal of Animal Sciences 92 (9): 1041–1045, September 2022/Article
https://doi.org/10.56093/ijans.v92i9.106568
Comparative analysis of intradermal tuberculin test and γ-interferon assay for
diagnosis of bovine tuberculosis
S S VYAVAHARE1, M K JHALA1 and B B BHANDERI1*
Anand Agricultural University, Anand, Gujarat 388 001 India
Received: 26 October 2020; Accepted: 21 July 2022
ABSTRACT
The present study was carried out in and around Anand district of Gujarat with the aim to assess the sensitivity
and specicity of IFN-γ assay as compared to intradermal tuberculin test for diagnosis of bovine tuberculosis during
the period 2011-12. The prevalence of bovine TB in the cattle was 26.19% by TST and 38.49% by IFN- γ assay.
Breed wise, highest prevalence of bovine TB was found in Gir (38.96%), followed by Triple cross (23.95%) and
Kankrej (16.45%) by TST; while by IFN- γ assay, highest prevalence was observed in Triple cross (42.70%). Age
wise, highest prevalence of bovine TB was found in adults (37.90%) followed by calves (26.66%) and heifers
(11.22%) by TST; while by IFN-γ assay, highest prevalence was observed in calves (66.66%) followed by adults
(44.35%) and heifers (22.44%). Sex wise, more prevalence of bovine TB was found in males (56.25%) than in
females (21.81%) by TST; while by IFN-γ assay, more prevalence was observed in females (39.25%) than in
males (31.25%). Sensitivity and specicity of TST in detecting bovine TB were 27.27% and 57.52% respectively,
compared to IFN-γ assay. ELISPOT assay showed 34.78% animals were found positive for bovine TB. IFN-γ assay
showed better sensitivity in detecting bovine TB at younger age compared to TST, and hence can be useful in timely
removal of the infected animals from the herd.
Keywords: Bovine tuberculosis, ELISPOT, IFN-γ, Tuberculin test
Present address: 1College of Veterinary Science and Animal
Husbandry, Anand Agricultural University, Anand, Gujarat.
*Corresponding author email: bbbhanderi@gmail.com
Bovine Tuberculosis (BTB) is a chronic bacterial
disease caused by Mycobacterium bovis, a member of
Mycobacterium tuberculosis complex involved in major
animal health problems with zoonotic and economic
implications (Dhaliwal et al. 2020). Infected animals are
main source of infection for other animals and, wildlife
species act as reservoirs of infection which presents a
complex epidemiological picture (El-Sayed et al. 2016).
The diagnostic tests currently approved and practiced for
BTB include the single intradermal tuberculin test (TST)
using puried protein derivative (PPD) antigens and/or
in vitro IFN-gamma release assay (IGRAs) (de Lisle et
al. 2017, Olea-Popleka et al. 2017 and Coad et al. 2019).
TST is widely used due to it’s cost effectiveness, easy
availability, long history of use, and lack of alternative
methods to detect BTB (Bezos et al. 2018). TST has many
limitations including difculties in administration and
interpretation of results, need for second visit to the farm,
low degree of standardization, and imperfect test accuracy
(Duignan et al. 2019). Use of crude PPD results in low
sensitivity and specicity of the test (Domenech et al.
2006, Casal et al. 2017).
Early secretory antigenic target-6 (ESAT-6) and
Culture Filtrate Protein-10 (CFP-10) are important TB
specic diagnostic target proteins in the whole blood
IFN-gamma assay (Praud et al. 2019, Srinivasan et al.
2019), due to their absence in many environmental, non-
tuberculous mycobacteria as well as in the BCG vaccine
strain (Mahairas et al. 1996). The IGRAs enzyme linked
immunospot (ELISPOT) assay was introduced in 1983
to detect cellular immunoglobulin production and then
adapted for measuring the cytokine response against a
dened antigen (Czerkinsky et al. 1983). ELISPOT is a
fundamental tool used in cellular immunology, providing
both qualitative and quantitative information on cellular
cytokine responses to dened antigens. ELISPOT assay
was approved and adopted as conrmatory diagnosis
in human latent TB (Lalvani et al. 2001). Present study
was carried on comparative diagnosis of BTB using TST,
IGRAs (IFN-γ) and ELISPOT in the cattle.
MATERIALS AND METHODS
A total of 252 cattle of three breeds, viz. Kankrej [n=79,
adult (39), heifer (35), calves (5)], Gir [n=77, adult (41),
heifer (25), calves (11)] and Triple cross [n=96, adult (44),
heifer (38), calves (14)] of various age groups who were
known to be TB infected during the previous regular annual
tuberculin skin testing were included (Supplementary Table 1).
TST: The tuberculin test was performed on the mid-neck
of the 252 cattle. Bovine puried protein derivative (2000
International Units, IVRI, Izatnagar) was used for TST.
The procedure and interpretation of TST was carried out as
SUPPLEMENTARY MATERIAL AVAILABLE ONLINE
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VYAVAHARE ET AL. [Indian Journal of Animal Sciences 92 (9)
1042
per the OIE Terrestrial Manual (2009).
IFN-γ assay: Whole blood stimulation assay was carried
out from the blood samples from 252 cattle within 5 h for
IFN-γ assay as per the method described by Bhavani et al.
(2011). Puried antigen ESAT-6 (5 μg/well) and CFP-10
(5 μg/well) were used (E. coli expressed Mycobacterium
bovis specic recombinant antigen codon optimized for
E. coli expression obtained from IIL, Hyderabad). After
stimulation, obtained plasma was stored at -20°C till the
IFN-γ assay was performed. IFN-γ assay is an enzyme linked
immunosorbent assay (ELISA) to measure bovine IFN-γ
derived from stimulated whole blood supernatant samples.
Following appropriate antigen-specic stimulation,
lymphocytes rapidly express and secrete cytokines. The
secreted-out cytokines from bovine cells were quantied
using the sandwich ELISA developed by IIL, Hyderabad.
IFN-γ assay was performed as per Schiller et al. (2010)
for the 252 blood supernatant samples obtained after whole
blood stimulation. Stimulation index was calculated as
optical density of antigen stimulation divided by optical
density of media. Stimulation index ≥ 2 is indicative of TB
+ve and < 2 is indicative of TB –ve.
ELISPOT assay: Blood samples from 23 animals
(Supplementary Table 1) were collected separately for
peripheral blood mononuclear cell (PBMC). PBMCs
separated from 23 animals and stored in liquid nitrogen
were processed for ELISPOT as per Veerasami et al.
(2011). The assays were performed in triplicate; results
were represented as a mean of triplicate wells and expressed
as spot forming cells (SFC) per million cells.
Statistical analysis: For statistical analysis of breed-
wise, age-wise and sex-wise prevalence of TB, results of
positivity obtained by both the tests, viz. TST and IFN-γ
assay were combined, to get the overall estimates of TB
prevalence. This was done as per the OIE guidelines. To
compare the breed-wise, age-wise and sex-wise proportion
of diseased animals chi-square test was carried out. The
level of chi-square test was 5% (carried as per Thruseld
et al. 2005)
RESULTS AND DISCUSSION
Tuberculin skin testing: Out of 252 animals, 66
(26.19%) animals were found to be positive by skin
testing. The positive reactors showed increase in skin
thickness with ≥ 4 mm along with diffused or extensive
oedema, exudation, necrosis, pain or inammation of
the lymphatic ducts and lymph nodes. Breed-wise, 13
(16.45%) animals of Kankrej (n= 79), 30 (38.96%) of Gir
(n=77) and 23 (23.95%) of Triple cross (n=96) breed were
positive by TST. Age-wise, 47 (37.90%) adults (n=124),
11 (11.22%) heifers (n=98) and 8 (26.66%) calves (n= 30)
were positive by TST. Sex-wise, 18 (56.25%) males (n=32)
and 48 (21.81%) females (n=220) were positive by TST
(Supplementary Table 2). Breed-wise, highest prevalence
was observed in Gir, followed by Triple cross and Kankrej.
Earlier, Ameni et al. (2006) reported that zebu cattle are
relatively resistant to TB infection than exotic cattle. Age-
wise, highest prevalence was observed in adults, followed
by calves and heifers. Regarding age-wise prevalence,
our results were in accordance with the previous study
reported by Linton and Dorshkind (2004) and Frasca and
Blomberg (2011). Sex-wise, more percentage of males
were positive than females by TST. Similar ndings for
sex-wise prevalence were previously reported by Kazwala
et al. (2001). Our study shows that prevalence was only
found in calves and heifers of Gir and Triple cross but not
of Kankrej by TST, indicating that the Kankrej animals are
relatively resistant to TB infection. Only adults of Kankrej
were found positive. The high prevalence in adult animals
possibly suggests their long-term exposure to the infection
and the declining immune defense with advancement of
age (Linton and Dorshkind 2004, Frasca et al. 2011). Our
study showed that Kankrej animals are relatively resistant
to TB infection compared to Gir and Triple cross.
IFN-γ assay: Out of 252 animals, 97 (38.49%) animals
were found positive by IFN-γ assay (indirect ELISA)
conducted after whole blood stimulation assay. If the
Stimulation index was ≥ 2, then the animal was considered
positive and if the stimulation index was < 2, then the
animal was considered negative. Breed-wise, 24 animals
(30.37%) of Kankrej (n=79), 32 (41.55%) of Gir (n=77)
and 41 (42.70%) of Triple cross (n=96) were positive by
IFN-γ assay. Age-wise, 55 (44.35%) adults (n=124), 22
(22.44%) heifers (n=98) and 20 (66.66%) calves (n=30)
were positive by IFN-γ assay. Sex-wise, 10 (31.25%)
males (n=32) and 87 (39.54%) females (n=220) were
found positive by IFN-γ test (Supplementary Table 3).
Breed-wise, highest prevalence was observed in Triple
cross, which was marginally higher than in Gir and lowest
in Kankrej by IFN-γ assay. Age-wise, highest prevalence
was observed in calves followed by adults and heifers. In
our study, IFN- γ assay was able to detect 12 more calves
and 11 more heifers than the TST, positive for TB infection
indicating that IFN-γ assay was able to detect TB infection
at an earlier age with better sensitivity. This may help in
taking proper preventive measures which can be taken to
reduce further progression of the disease. These ndings
were in accordance with the previous study reported by
Huda et al. (2003). Sex-wise, more prevalence was observed
in females as compared to males, which was contrary to the
TST results for sex-wise prevalence. It is difcult to explain
these contradictory ndings as the sample size for male
and female were very much different. Higher prevalence
of bovine TB in females was also observed by Nwanta
et al. (2011). Buddle et al. (2000) conducted a study to
evaluate a single mycobacterium antigen, ESAT-6 in the
IFN-γ test for use in skin test-positive cattle. The test based
Table 1. Number of the animal’s positive by TST and IFN-γ
assay
Positive
only by TST
Positive only
by IFN-γ
Positive by
both
Negative by
both
48 (19.04%) 79 (31.35%) 18 (7.15%) 107 (42.46%)
Total 252
14
COMPARATIVE ANALYSIS OF TST AND IFN γ September 2022] 1043
on ESAT-6 had a higher specicity than the test based on
PPD tuberculin, but this was offset by a small decrease in
sensitivity, while still maintaining a very high specicity.
In present study, two M. bovis specic antigens ESAT-6
and CFP-10 were used for improving the sensitivity and
specicity of the assay.
ELISPOT assay: Out of 252 animals, PBMCs of 23
animals were collected and stored in liquid nitrogen
until they were processed for ELISPOT. The assay was
performed in triplicates; results were represented as mean
of triplicate wells and expressed as spot forming cells
(SFC) per million cells. If 20 or greater than 20 spots
were recorded per well, then the animal was considered as
positive. If less than 20 spots were recorded per well, then
the animal was considered as negative (Supplementary
Fig. 1). The ELISPOT assay detects the number of IFN-γ
producing cells rather than the total amount of IFN-γ as is
measured by cytokine ELISA assays. ELISPOT has been
reported to be 10–200 times more sensitive in the detection
of cytokines than the ELISA (Parthsarthy et al. 2012). In the
present study, out of 23 animals, 8 (34.78%) animals were
found positive for bovine tuberculosis by ELISPOT assay
(Supplementary Fig. 1). Earlier, Zhang et al. (2009) tested
the hypothesis that the poor results of IFN-γ and ELISPOT
assays are due to lack of assay standardization, rather than
the inherent complexity of T-cell assays. They found that
ELISPOT assay provides reproducible results when the
assay procedure and data analysis were standardized. Their
study showed that ELISPOT assay is the ideal candidate for
the robust and reproducible monitoring of T- cell activity in
vivo, indicating ELISPOT assay as sensitive tool for the
diagnosis. Use of ELISPOT assay in HIV-AIDS vaccine
research was reported by Streeck et al. (2009). Vordermeir
et al. (2012) demonstrated that due to the exquisite
sensitivity of ELISPOT assay, it became an important tool
in vaccine development programme.
Comparative analysis of the tests employed: Sensitivity
and specicity of TST were assessed by comparing with
IFN-γ assay, as IFN-γ assay yielded more positive results
than the TST. Forty-eight (19.04%) and 79 (31.35%)
animals were found positive only by TST and IFN-γ assay,
respectively; 18 (7.15%) animals were found positive
by both TST and IFN-γ assay and 107 (42.46%) animals
were negative by both TST and IFN-γ assay (Table 1).
The sensitivity and specicity of TST was found 27.27%
and 57.52%, respectively with reference to IFN-γ assay,
whereas the overall agreement between both the tests was
49.60 % (Table 2). Overall comparison between TST and
IFN-γ assay revealed that out of 252 animals, 66 (26.19%)
were positive by TST and 97 (38.49%) by IFN-γ assay
(Supplementary Table 4). Out of 66 animals positive by
TST, 18 were positive while 48 were negative by IFN-γ
(Table 3). In our study, IFN-γ assay was able to detect 31
(12.30%) more animals positive for bovine TB indicating
that IFN-γ assay has more sensitivity than TST (Table 3).
Out of 124 adult animals, 47 (34.90%) and 55 (44.35%)
animals were positive by TST and IFN-γ assay, respectively.
The IFN-γ assay was able to detect TB infection in 8
(6.45%) more animals than TST. Out of 98 heifers, 11
(11.22%) animals were found positive by TST, and 22
(22.44%) animals were found positive by IFN-γ assay.
IFN-γ assay detected 11 (11.22%) more heifers positive for
TB infection (Supplementary Table 4). Out of 30 calves,
IFN-γ assay detected 20 (66.66%) calves which were
positive for TB infection. This shows that IFN-γ assay can
detect TB infection at earlier stage so that we can formulate
the measures to prevent further progression of disease.
On the contrary, TST was able to detect only 8 (26.66%)
calves positive for TB infection (Supplementary Table 4).
Looking at the comparison between TST and IFN-γ
assay, we can say that IFN-γ assay has more sensitivity
in detecting the TB infection as compared to TST. In our
study, IFN- γ assay was able to detect the TB infection at
earlier stage with better sensitivity. IFN-γ assay was in fact
able to detect a greater number of animals positive for TB
infection in each age group as compared to TST indicating
its higher sensitivity for detecting the infection.
Comparison between TST, IFN-γ and ELISPOT assay:
Out of 23 animals subjected for ELISPOT, 8, 3 and 7 were
positive by ELISPOT, TST and IFN-γ respectively. Three of
Table 2. Sensitivity and specicity analysis of TST for diagnosis of bovine TB with reference to IFN-γ assay
Test to be
compared
(TST)
Standard test (IFN-γ assay) Total Sensitivity Specicity Overall
agreement
Positive Negative
Positive 18 79 97
27.27 % 57.52 % 49.60 %Negative 48 107 155
Total 66 186 252
Table 3. Details of the animals positive by TST and IFN-γ assay
Breed Adults (n=124) Heifers (n=98) Calves (n=30) Total
Only by
IFN-γ assay
Only by
TST
Both Only by
IFN-γ assay
Only by
TST
Both Only by
IFN-γ assay
Only
by TST
IFN-γ assay
(%)
TST (%)
Kankrej 12 07 06 04 — — 02 — 24 (30.37%) 13 (16.45%)
Gir 11 14 06 07 03 02 06 05 32 (41.55%) 30 (38.96%)
Triple cross 16 10 04 09 06 — 12 03 41 (42.70%) 23 (23.95%)
Total 39 31 16 20 09 02 20 08 97 (38.49%) 66 (26.79%)
15
VYAVAHARE ET AL. [Indian Journal of Animal Sciences 92 (9)
1044
the ELISPOT positive animals were negative by both TST
and IFN-γ, 3 were also positive by IFN-γ assay but negative
by TST, one animal was positive by TST but negative by
IFN-γ, while one animal was positive by all the three tests
(Supplementary Fig. 1). Although it is difcult to conclude
the ELISPOT results with the limited number of samples
processed, it is logical to imply that ELISPOT can detect
positive animals in more sensitive way. Limited number of
documentations available in literature on ELISPOT applied
for TB diagnosis also suggests its meaningful application
for this purpose, especially when TB is an important
zoonosis and has severe consequences with public health
point of view. Present study was attempted to standardize
and apply this advanced technique, and the results obtained
should encourage further studies involving large number
of samples.
Overall breed, age and sex-wise BTB prevalence by chi-
square test: The chi-square value for breed-wise prevalence
obtained was 16.72 (p value 0.0002), which indicates that
difference in the breed wise disease prevalence is highly
signicant. The result of this study indicates that the Kankrej
animals are relatively resistant to TB infection compared to
Gir and Triple cross animals. This endorses the nding of
Ameni et al. (2006). The chi-square value obtained between
adults and calves was 7.223 (p value 0.0072) which indicates
that difference in age-wise prevalence was signicant. The
chi-square value obtained between adults and heifers was
31.248 (p value<0.0001) which indicates that difference
in age-wise prevalence was signicant. The chi-square
value obtained between calves and heifers was 35.191
(p value-<0.0001) which indicates that difference in age-
wise prevalence was signicant (Supplementary Table 5).
Our study showed that heifers are relatively resistant to
TB infection compared to calves and adult. These results
agree with the fact that the immune system of the calves
is underdeveloped. Similarly, high prevalence in adult
animals suggests that immunocompetence declines with
the age as suggested by Linton and Dorshkind (2004) and
Frasca et al. (2011). The chi-square value for sex-wise
prevalence obtained was 1.885 (p value 0.1697) which
indicates that difference in sex-wise prevalence was not
signicant. Our results were in accordance with Kazwala
et al. (2001)
In conclusion, prevalence of bovine TB in the cattle
was 26.19% by TST and 38.49% by IFN-γ assay. Overall
prevalence by both these assays was 57.53%. ELISPOT
showed promising results for detecting bovine TB and
should be explored further involving large number of
samples.
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