Antigen-specific interferon-gamma release is ...

Veterinary Immunology and Immunopathology 201 (2018) 12–15

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Antigen-specific interferon-gamma release is decreased following the single intradermal comparative cervical skin test in African buffaloes (Syncerus caffer)

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C. Clarkea, D. Cooperb, W.J. Goosena, R. McFadyena,1, R.M. Warrena, P.D. van Heldena, ⁎ S.D.C Parsonsa, M.A. Millera, a

DST/NRF Centre of Excellence for Biomedical TB Research/SAMRC Centre for Tuberculosis Research/Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, P.O. Box 241, Cape Town, 8000, South Africa Ezemvelo KZN Wildlife, P.O. Box 25, Mtubatuba, 3935, South Africa

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A R T I C LE I N FO

A B S T R A C T

Keywords: African buffalo Bovigam® Bovine tuberculosis Interferon gamma Interferon gamma release assay Tuberculin skin test

Effective disease management of wildlife relies on the strategic application of ante-mortem diagnostic tests for early identification and removal of M. bovis-infected animals. To improve diagnostic performance, interferongamma release assays (IGRAs) are often used in conjunction with the tuberculin skin test (TST). Since buffaloes are major maintenance hosts of M. bovis, optimal application of bovine TB diagnostic tests are especially important. We aimed to determine whether the timing of blood collection relative to the TST has an influence on IFN-γ production and diagnostic outcome in African buffaloes. Release of IFN-γ in response to bovine purified protein derivative (PPD), avian PPD and PC-HP® and PC-EC® peptides was measured by Bovigam® and an inhouse IGRA in a group of Bovigam®-positive and – negative buffaloes at the time the TST was performed and three days later. There was significantly lower IFN-γ release in response to these antigens post-TST in Bovigam®positive buffaloes, but no significant changes in Bovigam®-negative buffaloes. Also, a significantly greater proportion of buffaloes were Bovigam®-positive prior to the TST than three days later. We therefore recommend that blood samples for use in IGRAs be collected prior to or at the time the TST is performed to facilitate the correct identification of greater numbers of IGRA-positive buffaloes.

1. Introduction Mycobacterium bovis is the main causative agent of bovine tuberculosis (bTB), a chronic infectious disease of ecological and economic concern in livestock and wildlife (Buddle et al., 2009; Renwick et al., 2007). In South Africa, African buffaloes (Syncerus caffer) are important maintenance hosts of M. bovis (Renwick et al., 2007). Therefore early and accurate detection and removal of M. bovis-infected buffaloes are crucial to reduce disease transmission and support disease management practices (Buddle et al., 2009). To optimise the detection of infected animals, interferon-gamma (IFN-γ) release assays (IGRAs) are often used in conjunction with the tuberculin skin test (TST) in cattle (Sinclair et al., 2016). However, many cattle studies have shown that in vitro IFN-γ production is affected by the administration of skin tests, causing either a boost or a drop in IFN-γ release (Palmer et al., 2006; Ryan et al., 2000). However, the effect of the timing of blood

collection for IGRAs relative to the administration of the TST has not been investigated in African buffaloes. This study aimed to compare IFN-γ production in response to Bovigam® antigens in buffaloes prior to and three days following administration of the single intradermal comparative cervical skin test (SICCT). Furthermore, we aimed to determine whether the IGRA test outcome is affected by the timing of the test relative to the SICCT. In addition, serum cortisol values at day 0 and day 3 were compared with IGRA results to determine whether there was an association with a stress response. A greater understanding of the optimal timing of blood collection for IGRA relative to the administration of SICCT may improve the diagnosis of M. bovis infection in African buffaloes.

⁎ Corresponding author at: Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, PO Box 241, Cape Town, 8000 South Africa. E-mail address: [email protected] (M.A. Miller). 1 Present address: Centre for Proteomic and Genomic Research, Cape Town, South Africa.

https://doi.org/10.1016/j.vetimm.2018.05.002 Received 26 February 2018; Received in revised form 30 April 2018; Accepted 7 May 2018 0165-2427/ © 2018 Elsevier B.V. All rights reserved.


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2. Materials and methods

with cytokine assay results. The mean OD value of the negative control, included in the kit, was subtracted from the OD value of each serum sample and standard curve dilution. The cortisol concentrations were then determined from the standard curve. The cortisol ELISA was performed on 14 buffaloes that showed the greatest individual increase in IFN-γ release (group A) from pre- to post-SICCT and 18 animals that showed the greatest individual decrease in IFN-γ release (group B), as measured in the Bovigam® ELISA. The number of samples used in the assay was subject to the availability of anti-cortisol IgG coated ELISA wells.

2.1. Animals and SICCTs In this study, 286 African buffaloes were captured as part of an annual bTB control program in the Hluhluwe-iMfolozi Park (HiP), South Africa, and held in temporary bomas for 24 h–48 h before being chemically immobilised and single intradermal comparative cervical skin tests performed, as previously described (Parsons et al., 2011). PreSICCT (day 0) was regarded as the time the purified protein derivative (PPD) injections were performed, whereas post-SICCT (day 3) was 3 days later. Heparinised whole blood was collected from each buffalo from the jugular vein prior to performing the SICCT on day 0 and 3 days later when immobilised to measure the SICCT. Ethical approval for the capture and testing of these animals was granted by the Stellenbosch University Animal Care and Use Committee (SU-ACUM13-00016).

2.5. Post-mortem examination, mycobacterial culture and speciation All Bovigam®-positive (PPD, PC-HP, or PC-EC) animals, according to the day 0 Bovigam® assay results, were euthanised and post-mortem examinations performed. Animals were inspected for visible lesions consistent with bTB. Retropharyngeal, parotid, mandibular, tracheobronchial, mediastinal and mesenteric lymph nodes and tonsils were collected for mycobacterial culture; those with no visible lesions were pooled by head, thoracic and abdominal regions. Tissue samples were processed for culture in Mycobacteria Growth Indicator Tubes (Becton Dickinson, NY, USA) and genetic speciation performed, as previously described (Goosen et al., 2014; Warren et al., 2006).

2.2. Blood stimulation and Bovigam® ELISAs Aliquots of heparinised whole blood (250 μl) were incubated with 25 μl phosphate buffered saline (PBS); 25 μl bovine PPD (bPPD; final assay concentration 300 IU/ml); 25 μl avian PPD (aPPD; final assay concentration 300 IU/ml); 25 μl Bovigam® PC-HP peptide solution; 25 μl Bovigam® PC-EC peptide solution (All Prionics; Schlieren, Switzerland); and 25 μl pokeweed mitogen (Sigma-Aldrich, St. Louis, Missouri, USA) solution in PBS (final concentration 5 μg/ml), respectively. Tubes were incubated at 37° C for 20 h, then centrifuged at 6000 × g for 10 min, and plasma harvested. Interferon-gamma levels were measured in plasma using the Bovigam® ELISA (Prionics), according to the manufacturer’s instructions. For each buffalo, the optical density (OD) values measured in samples incubated with aPPD (ODaPPD) and PBS (ODNil) were subtracted from OD values of samples stimulated with bPPD (ODbPPD). Test results were calculated according to the manufacturer’s instructions. Buffaloes that tested positive in any Bovigam® assay (group 1; n = 55) and a group of animals that were Bovigam® and SICCT-negative (group 2; n = 21) were immobilised on day 3 (post-SICCT) for blood collection, stimulation and measurement of IFN-γ, as described above.

2.6. Statistical analysis All data were tested for normality with the D'Agostino & Pearson omnibus normality test. The antigen-specific IFN-γ results, measured on day 0 and day 3, were compared using the Wilcoxon signed-rank test. The proportions of Bovigam® positive buffaloes were compared pre- and post-SICCT using McNemar’s test. Paired- and unpaired t-tests were performed to compare the cortisol levels between time points and between groups, respectively. Observed differences were regarded as statistically significant at p < 0.05. Data are presented as median and interquartile range (IQR). Analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc). 3. Results and discussion The release of IFN-γ in response to Bovigam® antigens was measured in plasma from whole blood collected at day 0; 55 of 286 buffaloes had positive Bovigam® results to PPD, PC-EC or PC-HP. Thirty-one of 55 (56%) Bovigam® positive culled buffaloes (group 1) were M. bovis-positive on culture, confirming infection in this group. Change in median IFN-γ concentrations in PPD-stimulated samples (ODbPPD – ODaPPD) decreased significantly (p < 0.0001) from 786 pg/ ml (IQR: 182 – 2050) at day 0 to 288 pg/ml (IQR: 80–797) at day 3, as measured with the Mabtech IFN-γ ELISA in all buffaloes that were Bovigam®-positive on day 0 (group 1; n = 55). Specifically, median IFN-γ concentration in response to bPPD (ODbPPD – ODNil) decreased significantly (p < 0.0001) from day 0 (903.7 pg/ml; IQR 191.2–2191) to day 3 (395.7 pg/ml; IQR 148.9–996.9), with no significant change in IFN-γ concentration in response to aPPD (ODaPPD – ODNil) (79.5 pg/ml, IQR 37.58–204, at day 0; 71.6 pg/ml, IQR 29.4–158, at day 3), indicating that the differential decrease was not caused by environmental mycobacteria. Median IFN-γ concentration in response to PC-HP (ODHP – ODNil) decreased significantly (p < 0.0001) from 559 pg/ml (IQR: 97–1628) at day 0 to 171 pg/ml (IQR: 39–371) at day 3. Using the Bovigam® ELISA, median OD values of samples stimulated at day 0 were significantly higher (p < 0.0008) than at day 3 for differential responses to PPD (Fig. 1a), PC-HP (Fig. 1b), and PC-EC peptides (Fig. 1c) for group 1 buffaloes (n = 49). Specifically, the IFN-γ levels in response to bPPD (ODbPPD – ODNil) decreased significantly (p = .0008) from day 0 (OD: 0.9; IQR 0.22–1.9) to day 3 (OD: 0.5; IQR 0.15–0.96), but there was no significant change in IFN-γ levels in response to aPPD (ODaPPD – ODNil) (OD 0.08, IQR 0.03–0.2, at day 0; OD

2.3. Bovine IFN-γ ELISAs Absolute plasma IFN-γ concentrations in plasma samples were measured using a bovine ELISA, according to the manufacturer’s instructions (kit 3119-1H-20; Mabtech, Nacka Strand, Sweden). The sample OD of each well was measured using an LT 4000 Microplate Reader (Labtech, Uckfield, UK). The OD values were converted to IFN-γ concentrations based on a standard curve using serial dilutions of recombinant bovine IFN-γ proteins (0–1000 pg/ml), and calculated with GraphPad Prism 5 software (GraphPad Software, Inc. La Jolla, California, USA). For each buffalo, the antigen-specific IFN-γ concentration was calculated by subtracting the IFN-γ in the PBS (Nil) sample from that in the antigen-stimulated blood samples. Concentrations of IFN-γ were compared between the samples collected on day 0 and day 3. The number of animals tested in each assay was subject to the available volume of stimulated plasma. 2.4. Cortisol ELISA Whole blood was collected pre- and post-SICCT in serum vacutainer tubes (BD Pharmingen), as described above. Tubes were centrifuged at 3000 x g for 6 min and serum harvested for use in the cortisol ELISA. Blood cortisol levels were measured in pre- and post-SICCT serum samples, according to the manufacturer’s instructions, using a commercial competitive cortisol ELISA (Kit no. ab108665, Abcam, Cambridge, UK) to determine whether capture stress changed between the two sampling time points, and if there was a possible association 13


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Fig. 1. Antigen-specific IFN-γ release in response to a) PPD, b) PC-HP and c) PC-EC at day 0 and day 3 as measured by Bovigam® ELISA in plasma from buffaloes in groups 1 and 2. Horizontal bars represent medians. A diagnostic cut-off value of OD = 0.1 was used for the analysis of PC-HP and PC-EC.

0.06, IQR 0.02–0.19, at day 3). Furthermore, there were no significant changes (p > 0.05) in median IFN-γ levels in response to the Bovigam® antigens between the two time points for group 2 buffaloes, as measured on the Mabtech and Bovigam® (Fig. 1) ELISAs. These findings were similar to studies in cattle that reported a significant decrease in IFN-γ release following the skin test in response to bPPD, and ESAT-6 and CFP-10, i.e. M. bovis-specific antigens that occur in PC-HP® and PCEC® peptides (Whelan et al., 2004; Wood and Rothel, 1994). To determine whether the significant decrease in IFN-γ following the SICCT influenced the diagnostic outcome of the tests, the changes in proportions of all test-positive buffaloes (n = 55), using the Bovigam cut-off value, were calculated. A significantly greater proportion of group 1 buffaloes tested positive at day 0 compared to day 3 on the Bovigam® PPD (p = .046) and PC-EC assays (p = .004), but not with the Bovigam® PC-HP assay (p = .08) (Table 1). In group 2 buffaloes, no significant differences (p > 0.05) in the test outcomes between day 0 and day 3 were observed. Notably, day 3 test outcomes of group 2 showed that 1 of 21 (4.8%) buffaloes were Bovigam® PPD positive, none were PC-HP positive and 2 of 21 (9.5%) buffaloes were PC-EC positive, although all buffaloes were test negative at day 0. This increased IFN-γ release may have been caused by the injection of PPD for the SICCT, which agrees with other studies that showed that uninfected cattle may

Table 1 Proportions of test-positive buffaloes from group 1 at day 0 and day 3 as determined by Bovigam® PPD, PC-EC and PC-HP assays. Statistical change between time points for each Bovigam® assay is indicated by a p-value. Bovigam® assay

Day 0 Proportion (+)

Day 3 Proportion (+)

Change in proportion (+) from day 0 to 3

PPD PC-EC PC-HP

40/48 (83%) 38/49 (78%) 35/48 (73%)

33/48 (69%) 25/49 (51%) 27/48 (56%)

p = .046a p = .004a P = .08

a

Significant change in proportion positive buffaloes from day 0 to day 3.

show a slight boost in IFN-γ following the TST, especially those sensitised to environmental mycobacteria (Schiller et al., 2010). Unfortunately, as a limitation to this study, buffaloes were not culled based on the Bovigam® results generated on day 3 and therefore the bTB status of these animals could not be confirmed. Although this study aimed to investigate the effect of timing of blood collection for IGRA relative to the SICCT, many other factors may have contributed to the change in IFN-γ release following the SICCT. Since this study was performed on free-ranging buffaloes, we hypothesised that stress factors affecting these animals, caused by capture 14


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Conflict of interest There is no conflict of interest to be declared by the authors regarding this publication. Funding This work was supported by the South African Medical Research Council and National Research Foundation of South Africa (grant #86949). The content is the sole responsibility of the authors and does not necessarily represent the official views of the South African Medical Research Council or National Research Foundation. Acknowledgements Fig. 2. Cortisol concentration (ng/ml) as measured by a commercial competitive ELISA at day 0 and day 3 for buffaloes with an increase (Group A; n = 18) and a decrease (Group B; n = 18) in individual IFN-γ production on the Bovigam® assay. Horizontal bars represent means.

The authors acknowledge the assistance of Warren and Alicia McCall of the State Veterinary Services, Dr. Birgit Eggers, and staff of the Game Capture Unit of Ezemvelo KZN-Wildlife. In addition, we thank Marianna de Kock and Claudia Spies, Division of Molecular Biology and Human Genetics at Stellenbosch University, for their assistance with tissue cultures. For statistical advice, we thank Dr Francisco Olea-Popelka from Colorado State University.

and confinement, may have influenced our results. Studies have shown that stress causes a release of glucocorticoids, such as cortisol, which may lead to immune suppression and result in significantly reduced tuberculin reactions and IFN-γ release in cattle (Gormley et al., 2006). However, our results showed that there were no significant changes in cortisol levels between pre- and post-SICCT (Fig. 2). The mean cortisol concentrations of group A were 540 ng/ml (standard deviation, SD: 439 ng/ml) at day 0 and 596 (SD: 471 ng/ml) at day 3, whereas the mean cortisol concentrations of group B at day 0 and day 3 were 759 ng/ml (SD: 693 ng/ml) and 599 ng/ml (SD: 563 ng/ml), respectively. Nevertheless, another study has shown that immune suppression detected just after capture can continue for more than five weeks in zebras (Franceschini et al., 2008). This suggests that capture and confinement may have extended effects on immune responses in free-ranging buffaloes that are immobilised for TB testing. Cortisol concentrations in our study may have already increased by the time of the preSICCT measurement and remained elevated post-SICCT. Chemical immobilisation of buffaloes was performed using etorphine hydrochloride, an opioid chemically related to morphine. Studies have shown that morphine may result in inhibition of immune responses (Sacerdote, 2006), which may have contributed to a decline in IFN-γ release. However, the buffaloes were darted at both sampling sessions; therefore it is unlikely that the opioids caused the decrease in IFN-γ that was seen post-SICCT, since both time points would have been influenced by drug administration. However, buffaloes were treated with opioid antagonists (naltrexone or diprenorphine) after the first immobilisation, but not before the second blood collection event. In summary, to our knowledge this is the first study that has investigated the effect of timing of blood collection for IFN-γ release assays to screen herds for bTB, relative to the SICCT, in African buffaloes. Our results demonstrate that IFN-γ release in response to specific (PCHP and PC-EC) and sensitive antigens (PPD) was reduced post-SICCT in Bovigam®-positive animals, but remained unchanged in negative animals. The proportion of IFN-γ test-positive buffaloes was significantly less three days after administration of intradermal tuberculin for the SICCT in the Bovigam®-positive animals. In a herd with high M. bovis infection rates, effective bTB management efforts require removal of as many infected buffaloes as possible. Based on our results, we recommend that blood samples be collected for IFN-γ cytokine release assays on the day the SICCT is performed, rather than 3 days later, since significantly greater numbers of infected animals are detected preSICCT.

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