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

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.


INTRODUCTION
In Ethiopia, intensive dairy farms that raise genetically improved dairy cows have been established around cities and towns (Ameni et al., 2018;Mekonnen et al., 2019) to address national nutritional needs.
The development of the dairy sector has been constrained by the emergence of diseases associated with intensification, including bovine tuberculosis (bTB) (Ameni et al., 2007;Firdissa et al., 2012). bTB is an endemic disease in Ethiopia (reviewed by Sibhat et al., 2017) and the disease is affecting livestock production through reduction of productivity and trade restrictions (OIE, 2018;Tschopp et al., 2012). As a zoonotic disease, bTB poses a public health threat, especially in lowand middle-income countries (LMICs) like in Ethiopia (Ashford et al., 2001).
Many developed countries have controlled bTB in their livestock populations using detection and slaughter of reactor animals (Buddle et al., 2013;Schiller et al., 2010). However, in many LMICs, the implementation of such control is economically and societally unacceptable and different control strategies need to be considered. Vaccination is an alternative control strategy and BCG is the only currently available vaccine. Research trials evaluating the efficacy of BCG vaccination against M. bovis infection in cattle have demonstrated promising results (Vordermeier et al., 2009;Ameni et al., 2010;Ameni et al., 2018;Vordermeier et al., 2016a ; reviewed by Buddle et al., 2018). However, BCG vaccination sensitizes vaccinated animals to react to tuberculinbased tests, such as single intradermal cervical comparative tuberculin (SICCT) or single intradermal tuberculin (SIT) tests (Vordermeier et al., 2001;Whelan et al., 2010) as BCG was originally derived from M. bovis, compromising their specificity. The current standard for diagnosis of TB in cattle measures cell-mediated immune response to intradermal injection of tuberculin. The SIT test is performed by injecting 0.1 ml of 3000 IU of bovine purified tuberculin (PPD-B) into the skin of the midcervical region or in the base of the tail, while the SICCT test entails simultaneous injection of both PPD-B and avian PPD (PPD-A) side-byside into the skin of the neck for discriminating animals infected with M. bovis and those sensitized with M. avium complex or environmental non-tuberculous mycobacteria (reviewed by de la Rua-Domenech et al., 2006). However, both PPD-B and PPD-A are poorly defined antigens resulting in suboptimal sensitivity and inconsistent performance (Bezos et al., 2014;Buddle et al., 2009;Schiller et al., 2010). Therefore, there is a need for a diagnostic test that differentiates M. bovis infected from BCG-vaccinated animals (DIVA role).
Significant progress has been made applying defined antigens as DIVA tests for cattle (Jones et al., 2012;Srinivasan et al., 2019;Vordermeier et al., 1999;Vordermeier et al., 2002;Vordermeier et al., 2011;Vordermeier et al., 2016b), using mycobacterial antigens present in field strains of M. bovis but absent in the BCG vaccine. These antigens include early secretory antigen target-6 kDa (ESAT-6) and culture filtrate protein-10 kDa (CFP-10) (Pollock & Anderson, 1997;Vordermeier et al., 1999;Vordermeier et al., 2001). Their encoding genes are located within the region of difference 1 (RD1) of the M. bovis genome, a region was deleted from all BCG strains (Garnier et al., 2003;Gordon et al., 1999). As a result, T-cells of the BCG-vaccinated and/or noninfected cattle do not recognize ESAT-6 and CFP-10. However, the use of the cocktail of these two antigens showed a lower capacity in detecting infected animals compared with tuberculin-based tests (Sidders et al., 2008;Vordermeier et al., 2011). In attempts to overcome this limitation, the antigen Rv3615c was discovered to be a useful additional DIVA antigen to complement ESAT-6 and CFP-10 (Sidders et al., 2008).
This protein cocktail of Rv3615c, ESAT-6 and CFP-10 has previously been evaluated as a blood and skin test reagent, mainly in Bos taurus breeds, such as Holstein-Friesians (Casal et al., 2012;Vordermeier et al., 2016b) but not in zebu cattle or cross-breeds between zebus and taurine cattle. In the present study, the protein cocktailbased DIVA skin test (DSTc), as well as a fusion protein (DSTf) of the same antigens, were evaluated in Zebu-Holstein-Friesian crossbreed cattle under field condition.

Study animals and husbandry
The study was conducted on Holstein-Friesian x Zebu crossbred calves and cows. The calves were all male and recruited from bTB free dairy farms within 2 weeks of age. Upon arrival at our animal facility, the calves were screened by the whole blood interferon-gamma release assay (IGRA) to demonstrate freedom from infection (data not shown).
The cows (herein known as TB reactors) were recruited from a bTBpositive herd and tested positive for bTB upon recruitment by both IGRA and SICCT. The naïve calves and the TB reactor cows were kept in separate barns at the National Animal Health Diagnostic and Investigation Center at the Sebeta, Ethiopia. The calves were fed on pasteurized partially skimmed milk, hay and concentrate. The TB reactor cows were fed on hay and concentrate. Both the calves and the TB reactor cows were watered ad libitum. Hence, these 30 TB reactor cows were used for comparison of the DSTf with the DSTc and SICCT test; they were tested simultaneously with intradermal injection of DSTf and DSTc on one side of the neck, while SICCT was applied on the other side of the neck of the study animals.

Antigens
The 100 μg/ml of each protein (300 μg total protein/ml). The DSTc solution was stored at −80 • C until needed. The DSTf reagent was produced by Lionex following the same approach, with the exception that it was supplied as solution (300 μg total protein/ml) after buffer exchange against phosphate buffered saline (PBS; pH 7.4) and stored at 4 • C until being used.

Protein-based DIVA skin test
All study animals (BCG vaccinated and control calves; bTB-positive cows) were injected intradermally with 0.1 ml DSTc (30 μg total protein per dose) into the middle of the right side of the neck. For the comparison between the DSTc and DSTf, 0.1 ml DSTc (30 μg protein per dose) was injected 10 cm below the crest and 0.1 ml DSTf was injected 12 cm below DSTc on a vertical line. Skin thicknesses were measured before inoculation and at 72 h post inoculation. The measurements were done by the same operator using the same digital caliper in every testing.
Results are expressed as the difference in skin-fold thickness (in millimetre) before administration of the antigens and 72 h post administration. Skin reaction was considered positive if the increase in skin thickness at the DSTc or DSTf site was greater than or equal to 2 mm (Casal et al., 2012;Vordermeier et al., 2016b).

Single intradermal cervical comparative tuberculin test
The SICCT test was performed on the left side of the study animals in the middle of the neck. After preparation of the injection site, 0.1 ml PPD-A (3000IU/ml; Prionics, Lelystad, The Netherlands) was inoculated 10 cm below the crest and the same volume of PPD-B (2500IU/ml; Prionics) was injected at a site 12 cm apart from PPD-A injection site in vertical line in reactor cows. The skin thicknesses were measured just before injection and at 72 h post injection by the same operator using the same digital caliper, and the results were presented as change in skin thickness (mm) between the two readings. In case of the SIT test, skin reaction was defined as positive when the increase of skin thickness at PPD-B site was greater than or equal to 4 mm, otherwise considered as negative. For the SICCT test, the differences in the increase of skin thickness at the bovine and avian PPD injection sites were considered. An animal was considered to be positive when the increase in skin thickness at the bovine PPD site was greater than the increase in skin thickness at the site of the avian injection by at least 4 mm. If the differential increases between the two sites were equal to or less than 1 mm, or between 1 and 4 mm, the animal was considered negative or doubtful, respectively (OIE, 2018).

Data analysis
Data analysis was performed using Prism 8 (GraphPad Software). The skin-fold thickness increase was summarized using median and 95% confidence interval of median (95% CI) after assessment of normality of the data. Wilcoxon matched-pair signed rank test was performed for comparison of skin reactions induced by two defined antigens while

Diagnostic performance of DSTc in bTB-infected cows
The diagnostic sensitivity of the DSTc was tested in 68 naturally infected TB reactor cows from a confirmed bTB-positive herd (Data S1). The recorded median of skin thickness increase was 6.25 mm (95% CI = 5.30−6.76) at the DSTc injection site, while the median skin thicknesses were 10.82 mm (95% CI = 9.55−13.63) at PPD-B and 4.48 mm (95% CI = 3.59-5.03) at PPD-A sites. The result indicated that the median increase of skin thickness to DSTc injection was significantly lower (p < .001) than the median in skin thickness at the injection site of PPD-B (Figure 1c). On the other hand, by considering the SICCT results, the median of the differences in skin thickness at the injection site of PPD-B and the injection site at PPD-A was 7.10 mm (95% CI = 5.54−8.40), which did not statistically (p > .05) differ from the TA B L E 1 Comparison of the diagnostic performance of the cocktail protein-based DIVA skin test (DSTc) with the performances of the single intradermal tuberculin (SIT) test and the single comparative cervical tuberculin (SICCT) test in detecting bTB infection

Diagnostic performance of the DST fusion protein in reactor cattle
The diagnostic performance of the DSTf was evaluated in 30 TB reactor cows. The reactivity of the skin to injection with DSTf was compared with the skin reactivity after injection with DSTc, PPD-B and PPD-A.
The results of this experiment are presented in Figure 2

DISCUSSION
The present study was conducted to evaluate the performance of a  (Casal et al., 2012;Jones et al., 2012;Vordermeier et al., 2016a;Whelan et al., 2010 , b). However, this is the first study to evaluate the DSTc and DSTf proteins in zebu-taurine crossbred cattle in the context of developing countries; hitherto, only a peptide cocktail of these antigens has been tested in TB reactor cattle in Ethiopia (Srinivasan et al., 2019).
The relative specificity of the DSTc was evaluated in 35 non-infected and BCG-vaccinated calves, which were recruited from known bTB free dairy herds and also re-affirmed to be free of bTB by IGRA. Six weeks after subcutaneous inoculation with 1x10 6 CFU dose of BCG, they did not react to intradermal injection of the DSTc, giving a relative specificity of 100% (95% CI = 90.0-100). Similar to the present result, previous studies reported that antigenic protein-or peptidebased intradermal DSTc did not induce detectable skin reactions in BCG-vaccinated taurine cattle (Jones et al., 2012;Vordermeier et al., 2016b;Whelan et al., 2010). Similarly, all the 39 non-vaccinated calves did not react to all the three (DSTc, SIT and SICCT) tests and the speci-  (Jones et al., 2012;Srinivasan et al., 2019;Whelan et al., 2010). The stronger skin reaction to PPD-B could be because it consists of a more diverse range of immunogenic proteins (Borsuk et al., 2009), whereas the DSTc contains only the three mycobacterial proteins ESAT-6, CFP-10 and Rv3615c. Moreover, a dose of tuberculin solution contains greater protein content than a dose of the DSTc (Yang et al., 2013). The strong skin reaction following PPD-B could also be due to less purification compared to the highly purified DSTc. Except Casal et al. (2012), who recorded comparable medians skin thicknesses by injection of DSTc and PPD-B, other researchers recommended the possibility of strengthening the skin reaction to DSTc by adding Rv3020 (Jones et al., 2012;Vordermeier et al., 2016b).
In most of the earlier studies, the experiments evaluating DIVA tests were conducted on whole blood-based IFN-γ assays for easiness of the protocol to accommodate modifications (Vordermeier et al., 2016b).
However, the use of these DIVA reagents in the IFN-γ assay will be difficult to implement in areas with economic and technical constraints (Ameni et al., 2000). In contrast, the DIVA skin testing is a simpler technique and can easily be applied in the field in the same way as the tuberculin skin test. Thus, a recombinant fusion protein containing ESAT-6, CFP-10 and Rv3615-c (DSTf) was produced in a similar presentation as PPD-B, containing 1.2 ml (12 doses

CONCLUSION
This is the first study to investigate the performance of the DIVA skin test based on a cocktail/fusion protein of three mycobacterial antigens (ESAT-6, CFP-10 and Rv3615c) in zebu-taurine crossbred cattle. The data showed high sensitivity of the DSTc in TB reactor cows and its high specificity in BCG-vaccinated bTB free calves after 6 weeks of BCG vaccination. The data generated by the two DST preparations (cocktail and fusion) were comparable. Thus, the findings of this study demonstrated the potential utility of DSTc or DSTf to support BCG vaccinebased bTB control policies, although additional extended field evaluation of these tests is important for re-affirmation of the observations of this study.