Animal TB Research Group

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Sven Parsons, Senior Researcher, the research program embraces a multi-prong approach to animal TB.

mycobacterial organisms in animals novel biomarkers of TB in animals discovery of unique pathogenic mycobacteria of animals impact of TB at animal-human interfaces Our research incorporates a

1 1ANIMAL TB RESEARCH GROUP STELLENBOSCH UNIVERSITY Animal TB Research Group Stellenbosch University NEWSLETTER IN THIS ISSUE Animal TB Research Group December 2015 The Stellenbosch University Animal TB Research Group was formed in January Under the leadership of Professor Michele Miller (NRF South African Research Chair in Animal TB) and Dr. Sven Parsons, Senior Researcher, the research program embraces a multi-prong approach to animal TB. Areas of focus include: species-specific aspects of disease pathogenesis mycobacteria-specific immune responses and application to diagnostic techniques epidemiology and disease impact of mycobacterial organisms in animals novel biomarkers of TB in animals discovery of unique pathogenic mycobacteria of animals impact of TB at animal-human interfaces Our research incorporates a continuum from basic to applied science. Contributions of Collaborators Science functions best when performed in a spirit of collaboration and constructive communication. Our research group would be unable to achieve our objectives without the support of the many organizations and individuals that participate in a multitude of ways including providing access to animals and samples, contributing ideas, technical expertise, time and financial support to our projects. We wish to thank the following as well as many others not listed: South African National Parks (especially Dr. Markus Hofmeyr, Dr. Peter Buss, Marius Kruger, Jennifer Hofmeyr, Leana Rossouw, Guy Hausler, Milandie Kruger, and the entire VWS Capture Team) Dr. Dave Cooper and the Ezemvelo KwaZuluNatal Wildlife Team State Veterinary staff (especially Dr. Lin-Mari deklerk-lorist, Dr. Louis van Schalkwyk, animal health technicians of Kruger NP, Alicia and Warren McCall, Dr. Swellyn Davey) National Zoological Gardens of Pretoria staff (especially Dr. Emily Lane) University of Pretoria Faculty of Veterinary Science (especially Prof. Anita Michel, and Dr. Darshana Morar) Staff and students of the DST/NRF Centre of Excellence for Biomedical Tuberculosis Research/MRC Centre for TB Research, Stellenbosch University Meet the Team The Animal TB Research Group consists of senior scientists and a dynamic team of post-graduate students. Page 2 Research Update Get the latest updates from various Animal TB Research Group projects. Pages 2-5 Publication List Page 6

test development for cellmediated immune responses to M. bovis in buffalo and cattle Taime Olivier PhD student; research: characterization of immunological responses to M.

2 2 Meet the Team Academic Staff Michele A. Miller, DVM, MS, MPH, PhD Sven D.C. Parsons, BVSc, MSc, PhD Paul van Helden, PhD Eileen Hoal van Helden, PhD Louise Botha, MSc Students ( ) Wynand Goosen PhD student; research: investigation of novel biomarkers and diagnostic test development for cellmediated immune responses to M. bovis in buffalo and cattle Taime Olivier PhD student; research: characterization of immunological responses to M. bovis in African lions Ross McFadyen MSc student; research: humoral immune responses to M. bovis in buffalo Charlene Clarke Honour s (2014); MSc student; research: cell-mediated immune responses to M. suricattae in meerkats; investigation of temporal changes in immune responses to M. bovis in cattle and buffalo Eduard Roos MSc student; research: investigation of humoral and cell-mediated immune responses of warthogs to M. bovis Roxanne Higgitt Honour s student; research: investigation of antigen-specific immune responses in M. bovis exposed spotted hyenas ANIMAL TB RESEARCH GROUP WEBSITE For more information and details on our group s activities, visit our website: ences/molecular_biology_human_genetics/an imaltb Team in Hluhluwe-iMfolozi Game Park (HiP) acquiring samples for buffalo TB testing (Photo credit: Birgit Eggers) Evaluating Biomarkers for Diagnosis of BTB in Buffaloes The diagnosis of bovine tuberculosis in buffaloes currently relies on detection of immune responses, whether in vivo (tuberculin skin test, TST) or in vitro (gamma interferon release assays). However, the antigens used in these tests, purified protein derivative (PPD), are not absolutely specific for Mycobacterium bovis. Our group is investigating the use of alternative antigens and host biomarkers to improve detection of BTB. Out of 283 buffaloes tested in HiP using Bovigam PPD, the new peptide gamma interferon assays (PC-EC, PC-HP), and TST. Bovigam PPD assays appeared to detect a greater number of buffalo (48, 17%) compared to skin test (43, 15%). In previous studies, the peptide assays have been shown to have greater specificity but decreased sensitivity. However, recent research (Goosen et al. 2015) suggests that the cytokine biomarker IP-10 improves sensitivity in the peptide assays (Table 1). Table 1. Number of PPD Bovigampositive buffaloes detected by peptide assays using IFN-ɣ or IP-10. No. (%) of positive buffaloes Assay IFN-ɣ IP-10 PC-EC 36 (75%) 40 (83%) PC-HP 37 (77%) 41 (85%) Further research into IP-10 as a diagnostic biomarker is underway.

3 Detection of TB in wildlife can be challenging due to the lack of validated diagnostic tools. Species can vary widely in the usefulness of different techniques.

TUBERCULIN SKIN TESTING WARTHOGS ENDOSCOPIC TRACHEAL WASH IN LIONS ImmImmIOSCOPIC TRACHEAL WASH IN LIONS Humoral Immune Responses to Mycobacterium bovis in Wildlife by Antibody Responses in Buffaloes

appearing later in the course of infection. This suggests that serological diagnostic tests may be useful in identifying animals in more advanced stages of disease.

system. When sera from BTB positive buffalo were tested using the PPD-ELISA and commercially available VetTB DPP assay (Chembio), a significant correlation was observed in antibody responses.

Antibody Responses in Warthogs Warthogs have been considered a potential maintenance host for bovine tuberculosis; however there have been very few reports that provide information on screening and

3 3 Detection of TB in wildlife can be challenging due to the lack of validated diagnostic tools. Species can vary widely in the usefulness of different techniques. Some of the areas of current research are highlighted below. Dr. Sven Parsons prepares blood collection tubes for buffaloes. TUBERCULIN SKIN TESTING WARTHOGS ENDOSCOPIC TRACHEAL WASH IN LIONS ImmImmIOSCOPIC TRACHEAL WASH IN LIONS Humoral Immune Responses to Mycobacterium bovis in Wildlife by Antibody Responses in Buffaloes Immune responses to TB are characterized by a predominantly cell-mediated immune response during the early and intermediate stages of infection with a humoral (antibody-mediated) immune response appearing later in the course of infection. This suggests that serological diagnostic tests may be useful in identifying animals in more advanced stages of disease. In order to investigate serological tests for detection of bovine tuberculosis in buffalo, an inhouse PPD-ELISA was developed to detect antibodies in serum using a species-nonspecific detection system. When sera from BTB positive buffalo were tested using the PPD-ELISA and commercially available VetTB DPP assay (Chembio), a significant correlation was observed in antibody responses. These results suggest that buffalo can develop detectable antibodies which may be used as an ancillary diagnostic test for BTB. Antibody Responses in Warthogs Warthogs have been considered a potential maintenance host for bovine tuberculosis; however there have been very few reports that provide information on screening and detection of BTB, other than post-mortem. Between 2013 and 2015, 71 samples have been collected from Kruger National Park (and surrounding areas) and Mkuzi Game Reserve. Using the PPD-ELISA developed in-house for buffalo, commercial wild boar ELISA, and VetTB DPP, preliminary results suggest that warthogs develop a strong humoral response even in early stages of disease. Based on a cohort of warthogs with tissue culture results (MGIT), estimated sensitivity of PPD- ELISA was 87.5%, and 81.3% for the DPP assay. These findings indicate that screening of warthogs for BTB may be feasible using serological tests. Mandibular lymph node granuloma

4 Investigating M. bovis-specific Responses Using Gene Expression Assays Immune Responses in Spotted Hyenas Photo credit: Paul van Helden Table 2.

70 + P ND PvH7 5.30 7.07 - P + PvH8 0.15 1.84 - P + Few cases of mycobacterial disease have been reported in spotted hyena.

Phylogenetic analyses have revealed homology between chemokine genes, within the order Carnivora, which are important for responses to mycobacteria.

Table 2 shows results from 5 spotted hyena (KNP) using serological tests (Stat-Pak, DPP), TST, and gene expression scores from QFTstimulated whole blood.

00 - P + These preliminary data suggest that QFT tubes (Qiagen) may be useful in multiple species for inducing antigen-specific responses as measured by gene expression.

4 4 Investigating M. bovis-specific Responses Using Gene Expression Assays Immune Responses in Spotted Hyenas Photo credit: Paul van Helden Table 2. Gene expression assay score for CXCL8 and CXCL9 for hyena and concurrent TST, Stat-Pak, and DPP VetTB results. Animal CXCL8 CXCL9 TST Stat- Pak DPP VetTB PvH P ND PvH P ND PvH P + PvH P + Few cases of mycobacterial disease have been reported in spotted hyena. As predators/scavengers, they are potentially exposed to a wide range of pathogens. Therefore, there is an interest in characterizing immune responses in this species. Phylogenetic analyses have revealed homology between chemokine genes, within the order Carnivora, which are important for responses to mycobacteria. Using this information, relative gene expression (using qpcr) was determined in M. bovis-exposed spotted hyenas. Table 2 shows results from 5 spotted hyena (KNP) using serological tests (Stat-Pak, DPP), TST, and gene expression scores from QFTstimulated whole blood. Upregulation of CXCL9 expression was observed in response to TB antigen, especially in TST+ and Stat-Pak+ hyena (PvH4, PvH5). PvH P + These preliminary data suggest that QFT tubes (Qiagen) may be useful in multiple species for inducing antigen-specific responses as measured by gene expression. Cytokine Gene Expression for Detection of M. bovis-infected Lions Bovine tuberculosis can result in significant morbidity and mortality in infected lions. However, the only validated diagnostic test is the tuberculin skin test (TST), which requires two immobilization 72 hours apart. Blood-based tests such as gamma interferon release assays have also been described, although logistical constraints may limit potential use. Our research has investigated antigen-specific immune activation in lions by stimulating whole blood in QFT tubes, then storing and later extracting mrna for use in qpcr. Studies have shown that monokine-induced by gamma interferon (MIG; CXCL9 - gene) mrna is a more sensitive marker of immune response than IFN-ɣ. CXCL9 gene expression was significantly upregulated in response to TB peptide stimulation in M. bovis infected lions compared with uninfected lions. CXCL9 gene expression assays using QFT stimulated whole blood appear to be a sensitive method of detecting M. bovis infection in lions. Further work is underway to apply these techniques to larger populations. Photo credit: Peter Buss

5 OTHER RESEARCH DEVELOPMENTS Photo credit: Charlene Clarke TB in Meerkats TB in meerkats is caused by the novel Mycobacterium tuberculosis complex member, M. suricattae.

suricattae infection has resulted in the death of over 200 meerkats.

Supernatants from TB peptide stimulated whole blood have been tested in cytokine ELISA to detect immune activation.

Photo Photo credit: Lin-Mari deklerk-lorist Effect of Tuberculin Skin Test on IFN-ɣ Assays in Cattle and Buffaloes Tuberculin skin tests (TST) are currently the most commonly used TB screening tests

Our group has started to investigate the effect of TST on IGRA (Bovigam assay) in cattle and buffalo. Samples were obtained from a dairy cattle herd with a previous history of M. bovis infection.

Whole blood was incubated in QFT tubes pre- and post-tst (blood collected on d3). IFN-ɣ was measured in supernatants and showed a significant decrease in cytokine production post-tst.

5 5 OTHER RESEARCH DEVELOPMENTS Photo credit: Charlene Clarke TB in Meerkats TB in meerkats is caused by the novel Mycobacterium tuberculosis complex member, M. suricattae. Infection leads to generalized disease and rapid progression to death in clinically affected animals. This disease was first observed in the Kalahari Meerkat Project in Since that time, M. suricattae infection has resulted in the death of over 200 meerkats. In collaboration with researchers from the Royal Veterinary College (UK), immunoassays are being developed for detection of infection in KMP meerkats. Supernatants from TB peptide stimulated whole blood have been tested in cytokine ELISA to detect immune activation. IP- 10 ELISA optical density (OD) readings were significantly higher in KMP meerkats than in a colony of research meerkats. Photo Photo credit: Lin-Mari deklerk-lorist Effect of Tuberculin Skin Test on IFN-ɣ Assays in Cattle and Buffaloes Tuberculin skin tests (TST) are currently the most commonly used TB screening tests for cattle and buffaloes. However, previous studies have shown that TST may affect follow-up TST and cytokine assays, such as IFN-ɣ production. Our group has started to investigate the effect of TST on IGRA (Bovigam assay) in cattle and buffalo. Samples were obtained from a dairy cattle herd with a previous history of M. bovis infection. Whole blood from TST positive cattle was collected 6 and 41 days post-tst and incubated with PPDs (Bovigam). Buffaloes were tested using TST as part of a screening program in HiP (2015). Whole blood was incubated in QFT tubes pre- and post-tst (blood collected on d3). IFN-ɣ was measured in supernatants and showed a significant decrease in cytokine production post-tst. Antelope TB Sporadic cases of bovine tuberculosis has been reported in a variety of antelope, including greater kudu, bushbuck, and nyala. We are currently developing tools for characterizing the immune responses to M. bovis in these species. We are interested in obtaining information or samples from TB cases in any antelope species. Please feel free to contact us if you are interested in contributing to this research. TB Exposure in Working African Elephants M. tuberculosis and M. bovis have been reported in captive elephants worldwide. Close prolonged contact of elephants with infected handlers may increase the risk of anthropogenic transmission. In partnership with Prof. Anita Michel, Dr. Tapiwa Haniyre, Dr. Francisco Olea-Popelka, Dr. Chris Foggin and Jessica Dawson of Vic Falls Wildlife Trust, a project to evaluate risk of TB in captive elephants and handlers is in progress. Serological assays, cytokine production, and culture of trunk washes as well as risk factor survey evaluation will be used to assess infection status. Photo credit : Jessica Dawson Studies are currently investigating the use of this and other assays for diagnosing TB in meerkats ante-mortem.

6 6 Publications by Animal TB Research Group Miller, M., G. Fleming, S. Citino, and M. Hofmeyr Hippopotami. In West, G., D. Heard, and N. Cauklett (eds.), Zoo Animal & Wildlife Immobilization and Anesthesia, 2 nd ed. Wiley-Blackwell Publishing, Ames, Iowa. Pp Miller, M., and P. Buss. Rhinoceridae (Rhinoceroses) In Miller, R.E., and M.E. Fowler (eds.), Zoo and Wild Animal Medicine, Current Therapy 8 th ed., Elsevier, St. Louis, Missouri. Pp Landolfi, J.A., M. Miller, C. Maddox, F. Zuckermann, and K.A. Terio Differences in immune cell function between Mycobacterium spp. infected and uninfected Asian elephants. Tuberculosis 94: Goosen, W.J., M.A. Miller, N.N. Chegou, D. Cooper, R.M. Warren, P.D. van Helden, and S.D.C. Parsons Agreement between assays of cell-mediated immunity utilizing Mycobacterium bovis-specific antigens for the diagnosis of tuberculosis in African buffaloes (Syncerus caffer). Vet. Immunol. Immunopath. 160: Radcliffe, R.W., P. Morkel, M. Jago, A.A. Taft, P. dupreez, M.A. Miller, D. Candra, D.V. Nydam, J.S. Barry, and R.D. Gleed Pulmonary dead space in free-ranging immobilized black rhinoceroses (Diceros bicornis) in Namibia. J. Zoo Wildl. Med. 45(2): Haw, A., M. Hofmeyr, A. Fuller, P. Buss, M. Miller, G. Fleming, and L. Meyer Butorphanol with oxygen insufflation corrects etorphine-induced hypoxaemia in chemically immobilized white rhinoceros (Ceratotherium simum). BMC Vet. Res. 10: Goosen, W.J., D. Cooper, R.M. Warren, M.A. Miller, P.D. van Helden, and S.D.C. Parsons The evaluation of candidate biomarkers of cell-mediated immunity for the diagnosis of Mycobacterium bovis infection in African buffaloes (Syncerus caffer). Vet. Immun. Immunopath. 162: Miller, M., and R. Radcliffe Health. In Metrione L., and A. Eyres (eds.), Rhino Husbandry Manual, International Rhino Foundation, Fort Worth, Texas. Pp , , , Roth T., S. Shurter, M. Miller, M.A. Stoops, R.R. Swaisgood, M. Schlegel, E. Dierenfeld, and A. Eyres Research. In Metrione L., and A. Eyres (eds.), Rhino Husbandry Manual, International Rhino Foundation, Fort Worth, Texas. Pp , , , Miller, M., P. Buss, J. Hofmeyr, F. Olea- Popelka, S. Parsons, and P. van Helden Antemortem diagnosis of Mycobacterium bovis infection in freeranging African lions (Panthera leo) and Implications for Transmission. J. Wildl. Dis. 51: Miller, M.A., R. Greenwald, and K.P. Lyashchenko Potential for serodiagnosis of tuberculosis in black rhinoceros. J. Zoo Wildl. Med. 46: Landolfi, J.A., K.A. Terio, M. Miller, B.F. Junecko, and T. Reinhart Pulmonary tuberculosis in Asian elephants (Elephas maximus): histologic lesions with correlation to local immune responses. Vet. Pathol. 52: Buss, P., F. Olea-Popelka, L. Meyer, J. Hofmeyr, N. Mathebula, M. Kruger, A. Bruns, L. Martin, and M. Miller Evaluation of cardiorespiratory, blood gas, and lactate values during extended immobilization of white rhinoceros (Ceratotherium simum). J. Zoo Wildl. Med. 46: Goosen, W.J., D. Cooper, M.A. Miller, P.D. van Helden, and S.D.C. Parsons IP- 10 is a sensitive biomarker of antigen recognition in whole blood stimulation assays used for the diagnosis of Mycobacterium bovis infection in African buffaloes (Syncerus caffer). Clin. Vaccine Immunol. 22: doi: /cvi Haw, A., M. Hofmeyr, P. Buss, M. Miller, L. Meyer, A. Fuller, and G. Fleming Evaluation of butorphanol combined with oxygen insufflation to improve cardiorespiratory function during chemical immobilization of free-ranging white rhinoceros (Ceratotherium simum). J. S. Afr. Vet. Assoc. 86(1), Art.#1276, 10 pages Miller, M., P. Buss, F. Olea-Popelka, R. Wanty, S. Parsons, and P. van Helden Baseline hematologic parameters for free-ranging white rhinoceros (Ceratotherium simum) in Kruger National Park, South Africa. J. Wildl. Dis. doi: / Miller, M., and K. Lyashchenko Mycobacterial Infections in Other Zoo Animals. In Mukundan, H., M.A. Chambers, W.R. Waters, and M.H. Larsen (eds.), Tuberculosis, Leprosy and Mycobacterial Diseases of Man and Animals: The Many Hosts of Mycobacteria, CABI, Boston, Massachusetts. Pp Miller, M., P. Buss, L.-M. deklerk-lorist, J. Hofmeyr, G. Hausler, K. Lyashchenko, E.P. Lane, L. Botha, S. Parsons, and P. van Helden Application of Rapid Serological Tests for Detection of Mycobacterium bovis Infection in Free- Ranging Warthogs (Phacochoerus africanus) Implications for Ante-Mortem Disease Screening. J. Wildl. Dis. Manuscript accepted. 19. Miller, M., M. Kruger, M. Kruger, F. Olea- Popelka, and P. Buss A Scoring System to Improve Decision-Making and Outcomes in the Adaptation of Recently Captured White Rhinoceros (Ceratotherium simum) to Captivity. J. Wildl. Dis. Manuscript accepted. 20. Olivier, T.T., I.S.W.M. Viljoen, J. Hofmeyr, G.A. Hausler, W.J. Goosen, A. Tordiffe, P. Buss, A.G. Loxton, R.M. Warren, M.A. Miller, P..D. van Helden, and S.D.C. Parsons Development of a gene expression assay for the diagnosis of Mycobacterium bovis infection in African lions (Panthera leo). Transbound. Emerg. Dis. Manuscript accepted. 21. Clarke, C., P. van Helden, M. Miller, and S. Parsons. Animal-adapted members of the Mycobacterium tuberculosis complex endemic to the Southern African subregion. J. S. Afr. Vet. Assoc. Manuscript accepted. 22. Parsons, S.D.C., McGill, K., Doyle, M., Goosen, W., van Helden, P.V., Gormley, E. Antigen specific IP-10 release is a sensitive diagnostic biomarker of Mycobacterium bovis infection in cattle. Manuscript submitted.

7 7 Animal TB Research Group Stellenbosch University P.O. Box 241 Cape Town 8000 South Africa For more information about the Animal TB Research Group, please contact: Prof. Michele Miller, Dr. Sven Parsons,

Development and evaluation of a diagnostic cytokine-release assay for Mycobacterium suricattae infection in meerkats (Suricata suricatta)

Clarke et al. BMC Veterinary Research (2017) 13:2 DOI 10.1186/s12917-016-0927-x RESEARCH ARTICLE Open Access Development and evaluation of a diagnostic cytokine-release assay for Mycobacterium suricattae