Investigation of temporal changes in immune responses to Mycobacterium bovis in cattle and African buffaloes (Syncerus caffer).

Clarke, Charlene (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Cattle and African buffaloes are major maintenance hosts of Mycobacterium bovis in South Africa and therefore serve as a potential source of infection for other animals and humans. Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB). Identification and removal of M. bovis-infected animals from the herd at early stages of infection is important for effective disease management and relies on the strategic application of ante-mortem diagnostic tests. Ante-mortem diagnostic tests rely on the detection of specific immunological responses in the animal. However, these responses vary over the course of infection and therefore the accuracy of the diagnostic test may change, depending on the stage of infection. We therefore aimed to investigate the temporal changes in antigen-specific cytokine production and humoral responses to BTB in buffaloes. Interferon-gamma (IFN-ɣ) and IFN-ɣ-induced protein 10 (IP-10) production in response to mycobacterial antigens ESAT-6/CFP-10, and serum antibodies to bovine purified protein derivative (bPPD) were measured in a group of chronically test-positive, newly converted test-positive and test-negative buffaloes over one year. No consistent trends in immune responses over time were observed in any of the groups. Cytokine release assays (CRAs) are often used in conjunction with the tuberculin skin test (TST) to improve diagnostic performance. However, many studies have shown that the TST may influence IFN-ɣ production and thereby the outcome of immunodiagnostic tests in cattle. However, these results were conflicting. A better understanding of the influence of the TST on cytokine production and the duration of this effect in a South African situation is required. We therefore aimed to investigate if the TST has an influence on IFN-ɣ and IP-10 production and test outcome in cattle and African buffaloes. IFN-ɣ and IP-10 release were measured in response to bPPD, avian PPD, ESAT-6 and CFP-10 in groups of TST-positive cattle 6, 7, 21, 41 and 78 days post-TST and in a group of TST-negative cattle 7, 21 and 78 days post-TST. IFN-ɣ and IP-10 levels in response to PPD were elevated 1-3 weeks post-TST, followed by a decrease by 41 days, suggesting immune boosting by the TST. ESAT-6/CFP-10-specific cytokine release showed conflicting results, with a group of animals showing decreased cytokine production by 41 days, whereas another group showed no change in cytokine release over time. Our findings suggest that cattle should not be tested with CRAs between 6 and 41 days post-TST to avoid boosting of cytokine levels and inaccurate test results. IFN-ɣ and IP-10 release in response to PC-EC®, PC-HP®, bPPD and avian PPD were measured in a group of Bovigam®-positive and negative buffaloes at the time the TST was performed and three days later. In Bovigam®-positive buffaloes a significant decrease in cytokine production and in the proportion of test positive animals were observed three days post-TST in response to all antigens, except aPPD. Bovigam®-negative animals were not influenced by the TST. It is therefore recommended that buffaloes should be sampled pre-TST to identify all possible M. bovis-positive animals in this herd of high BTB incidence. Accuracy of diagnostic assays may be affected by many factors, including the immunological stage of M. bovis-infection, time interval between the performance of the TST and CRA, exposure to environmental mycobacteria and BTB incidence in the herd. The correct application and interpretation of diagnostic assays of high specificity and sensitivity is required to overcome some of these factors. Therefore, information of the BTB status of the herd, exposure to environmental mycobacteria, and an understanding of the aim of the disease management plan is required.

AFRIKAANSE OPSOMMING: Beeste en Afrika buffels is belangrike onderhouds gaste van Mycobacterium bovis in Suid Afrika en dien daarom as ‘n bron van infeksie vir ander diere. Hierdie patogeen is die oorsaak van bees tuberkulose (BTB) en indentifisering en verwydering van M. bovis-geïnfekteerde diere uit die kudde in ‘n vroeë stadium van infeksie is belangrik vir effektiewe siekte beheer. Dit is afhanklik van die strategiese aanwending van ante mortem diagnostiese toetse. Daarom, vir die verbetering van toets interpretasie, was ons doel om die temporale veranderinge in antigeen-spesifieke sitokien produksie en humorale reaksies tot BTB in buffels te ondersoek. Interferon-gamma (IFN-ɣ) en IFN-ɣ-geïnduseerde proteïen 10 (IP-10) produksie in reaksie tot mikobakteriële antigene ESAT-6/CFP-10, en teenliggaam vlakke was bepaal in ‘n groep chronies Bovigam®-positiewe (n = 5), pas omgeskakelde Bovigam®-positiewe (n = 5) en Bovigam®-negatiewe buffels (n = 8) oor ‘n 1 jaar typerk. Daar was geen spesifieke patrone in immuun reaksies oor tyd in enige van die groepe nie, alhoewel die chronies Bovigam®-positiewe diere ESAT-6/CFP-10 positief gebly het. ‘n Een jaar studie tydperk was nie genoeg om veranderinge in sitokien en teenliggaam vlakke in natuurlik-geïnfekteerde buffels te evalueer nie. IFN-ɣ vrystellings toetse (IGRAs) word algemeen saam met die tuberkulien vel toets (TST) gebruik om die diagnostiese prestasie te verbeter. Baie studies het egter bevind dat die TST ‘n effek op in vitro IFN-ɣ produksie in beeste het. Daarom was die doel van die studie om die effek van die TST op sitokien produksie en toets sensitiwiteit, spesifisiteit en resultaat in beeste en buffels te bepaal. Vrystelling van IFN-ɣ en IP-10 was bepaal in reaksie tot PPDs, ESAT-6 en CFP-10 in ‘n groep TST-positiewe beeste 6 en 41 dae na die TST (groep 1). Soortgelyk, was sitokien vrystelling in TST-positiewe en negatiewe beeste by 7, 21 en 78 dae na die TST (groep 2) bepaal. IFN-ɣ and IP-10 vlakke in reaksie tot PPD was verhoog 1-3 weke na die TST, gevolg deur ‘n afname teen 41 dae, wat dui op ‘n immunologiese “boosting” deur die TST. Sitokien produksie in respons tot ESAT-6/CFP-10 in group 1 het ook betekenisvol afgeneem. Sitokien “boosting” mag egter verband hou met blootstelling aan omgewings mikobakterieë in hierdie kudde met ‘n lae voorkoms van BTB. Ons resultate ondersteun die gebruik van toetse wat ESAT-6/CFP-10 gebruik om beeste rondom 41 dae na die TST te toets en so die akkuraatheid van die toets resultate te verbeter. IFN-ɣ en IP-10 vrystelling in respons tot PC-EC, PC-HP, bovine PPD en avian PPD was bepaal in ‘n groep Bovigam®-positiewe en negatiewe buffels op dieselfde tyd van die uitvoer van die TST en 3 dae later. Sitokien produksie en die proporsie toets-positiewe diere het betekenisvol afgeneem 3 dae na die TST in respons tot al die antigene, behalwe avian PPD, in Bovigam®-positiewe buffels. Dit word daarom aanbeveel dat bloedmonsters geneem moet word wanneer die TST uitgevoer word om al die TST-positiewe buffels te identifiseer. Akkuraatheid van diagnostiese toetse kan deur vele faktore geaffekteer word, insluitend die immunologiese stadium van M. bovis-infeksie, tyd interval tussen die TST en sitokien toetse, blootstelling aan omgewings mikobakterieë, en die BTB voorkoms in die kudde. Die korrekte aanwending en interpreatsie van diagnostiese toetse met hoë sensitiwiteit en spesifisiteit word benodig om sommige van die faktore te oorkom. Inligting van the BTB voorkoms in die kudde, en die blootstelling aan omgewings mikobakterieë asook ‘n begrip van die doel van die spesifieke siekte beheer plan is daarom belangrik.

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