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. 2022 Jul;69(4):e1-e9.
doi: 10.1111/tbed.14252. Epub 2021 Aug 19.

Field evaluation of specific mycobacterial protein-based skin test for the differentiation of Mycobacterium bovis-infected and Bacillus Calmette Guerin-vaccinated crossbred cattle in Ethiopia

Berecha Bayissa  1   2 , Asegedech Sirak  1   3 , Aboma Zewude  4 , Adane Worku  1 , Balako Gumi  1 , Stefan Berg  5 , R Glyn Hewinson  6 , James L N Wood  7 , Gareth J Jones  5 ; ETHICOBOTS consortium; H Martin Vordermeier  5   6 , Gobena Ameni  1   8
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Field evaluation of specific mycobacterial protein-based skin test for the differentiation of Mycobacterium bovis-infected and Bacillus Calmette Guerin-vaccinated crossbred cattle in Ethiopia

Berecha Bayissa et al. Transbound Emerg Dis. 2022 Jul.

Abstract

Bovine tuberculosis (bTB) challenges intensive dairy production in Ethiopia and implementation of the test and slaughter control strategy is not economically acceptable in the country. Vaccination of cattle with Bacillus Calmette-Guerin (BCG) could be an important adjunct to control, which would require a diagnostic test to differentiate Mycobacterium bovis (M. bovis)-infected and BCG-vaccinated animals (DIVA role). This study describes an evaluation of a DIVA skin test (DST) that is based on a cocktail (DSTc) or fusion (DSTf) of specific (ESAT-6, CFP-10 and Rv3615c) M. bovis proteins in Zebu-Holstein-Friesians crossbred cattle in Ethiopia. The study animals used were 74 calves (35 BCG vaccinated and 39 unvaccinated) aged less than 3 weeks at the start of experiment and 68 naturally infected 'TB reactor' cows. Six weeks after vaccination, the 74 calves were tested with the DSTc and the single intradermal cervical comparative tuberculin (SICCT) test. The TB reactor cows were tested with the DSTc and the SICCT test. Reactions to the DSTc were not observed in BCG-vaccinated and unvaccinated calves, while SICCT test reactions were detected in vaccinated calves. DSTc reactions were detected in 95.6% of the TB reactor cows and single intradermal tuberculin positive reactions were found in 98.2% (95% confidence interval, CI, 92.1-100%). The sensitivity of the DSTc was 95.6% (95% CI, 87.6-99.1%), and significantly (p < .001) higher than the sensitivity (75%, 95% CI, 63.0-84.7%) of the SICCT test at 4 mm cut-off. DSTf and DSTc reactions were correlated (r = 0.75; 95% CI = 0.53-0.88). In conclusion, the DSTc could differentiate M. bovis-infected from BCG-vaccinated cattle in Ethiopia. DST had higher sensitivity than the SICCT test. Hence, the DSTc could be used as a diagnostic tool for bTB if BCG vaccination is implemented for the control of bTB in Ethiopia and other countries.

Keywords: BCG vaccination; DIVA skin test; bovine tuberculosis; crossbred cattle; specific mycobacterial proteins.

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Conflict of interest statement

The authors declare that there are no competing interests.

Figures

FIGURE 1
FIGURE 1
Skin thickness in response to DSTc, PPD‐B, PPD‐A and PPD‐(B‐A) in 35 BCG‐vaccinated calves (Panel a), in 39 non‐vaccinated naïve calves (Panel b) and in 68 naturally infected reactor cattle (Panel c). Individual animal skin thickness change (millimetre) between the pre‐ and post‐skin test readings was represented by solid squares for DSTc, open circles for PPD (B‐A), solid triangles for PPD‐B and solid circles for PPD‐A with a horizontal line providing the median change of respective defined antigen. The statistical difference in skin reaction was determined using non‐parametric Friedman test with Dunn's multiple comparison test. There was a significant difference (p < .001) between DSTc and tuberculin in causing skin reaction in the BCG‐vaccinated calves, while similar skin reaction (p > .05) was developed following inoculation of all defined antigens in naïve calves. Significantly (p < .001) stronger skin response in naturally infected reactor cattle was also developed following PPD‐B intradermal injection compared to the outcome of DSTc. The dashed horizontal lines at 2 and 4 mm are the cut‐offs used for DSTc, and PPD‐(B‐A) and PPD‐B, respectively
FIGURE 2
FIGURE 2
Comparison of skin reaction response of DSTf to the response of PPD‐A, PPD‐B, PPD‐(B‐A) and that of DSTc in 30 naturally infected cows. The skin‐fold thickness was measured before and after 72 h of injections. Results are shown as increases in skin thickness (mm), which are represented by open square, solid square, open circle, solid triangle and solid circle with horizontal lines providing the median skin thickness change of respective antigen. PPD‐B induced significantly stronger skin reaction (p < .001) than that caused by DSTf using the Friedman test (repeated measures non‐parametric analysis of variance) with Dunn's multiple comparison tests. There was no significant difference (p > .05) between the responses induced by DSTf and DSTc in reactor cattle. The dashed horizontal lines at 2 and 4 mm are the cut‐offs used for DSTf and DSTc, and PPD (B‐A) and PPD‐B, respectively
FIGURE 3
FIGURE 3
Correlation of skin reaction responses between the DSTf (dose 30 μg) and the DSTc (dose 10 μg per protein). Solid black circle represents an individual study animal. The dashed horizontal and vertical lines at 2 and 4 mm are the cut‐offs used for DSTc and DSTf, respectively
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