Optimization of a whole blood gamma interferon assay for the detection of sheep infected with Mycobacterium avium subspecies paratuberculosis

Optimization of a whole blood gamma interferon assay for the detection of sheep infected with Mycobacterium avium subspecies paratuberculosis
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  See discussions, stats, and author profiles for this publication at: Optimization of a Whole Blood GammaInterferon Assay for the Detection of SheepInfected with Mycobacterium Avium...  Article   in  Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc · March 2010 DOI: 10.1177/104063871002200206 · Source: PubMed CITATIONS 5 READS 35 6 authors , including: Some of the authors of this publication are also working on these related projects: Genomic Selection and Advanced Prawn Genomics and Breeding   View projectStrengthening food and nutrition security through family poultry and crop integration in Tanzania andZambia   View projectKatrina L BoswardUniversity of Sydney 42   PUBLICATIONS   316   CITATIONS   SEE PROFILE Peter C ThomsonUniversity of Sydney 292   PUBLICATIONS   2,191   CITATIONS   SEE PROFILE David EmeryUniversity of Sydney 129   PUBLICATIONS   2,550   CITATIONS   SEE PROFILE Richard J WhittingtonUniversity of Sydney 367   PUBLICATIONS   6,616   CITATIONS   SEE PROFILE All content following this page was uploaded by Peter C Thomson on 09 January 2017. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  Optimization of a whole blood gamma interferon assay for thedetection of sheep infected with  Mycobacterium avium subspecies  paratuberculosis Katrina L. Bosward, 1 Navneet K. Dhand, Douglas J. Begg, Peter C. Thomson, David L. Emery,Richard J. Whittington Abstract.  The capacity of a commercially available gamma interferon (IFN c ) assay to detect infectedsheep early in the pathogenesis of Johne’s disease enables the removal of such animals from the flock beforebacterial shedding and pasture contamination. However, nonspecific IFN c  responses in the assay have meantthat to achieve high-test specificity, there has been a reduction in sensitivity. Although the optimal conditionsfor the use of the assay in cattle have been well documented, there have been few studies optimizing the assayfor use in sheep. The current study details the effect of anticoagulant, duration of incubation, cellconcentration, blood storage temperature, time of stimulation of cells with antigen relative to time of samplecollection, and temperatures during transit on IFN c  synthesis. Maximal IFN c  synthesis occurred withincubation periods of 48 hr in blood collected into heparinized tubes. Decreasing the leukocyte population bydiluting the total peripheral blood leukocyte concentration was associated with a decreasing IFN c  response.Conversely, concentrating the peripheral blood concentration 2-fold resulted in an increase in the IFN c production. In field studies, immediate incubation of blood samples with antigen at 37 u C resulted in largerIFN c  responses; however, significantly lower IFN c  values were obtained if the samples were transported atambient temperature. The results of this study indicate that optimization of the IFN c  assay may enableincreased synthesis of IFN c  during the stimulation phase of the assay and that future work may determinewhether this translates to increased sensitivity of the assay in detecting early infections in sheep. Key words:  Bovigam assay; gamma interferon; Johne’s disease; paratuberculosis; sheep. Introduction The Bovigam sandwich assay a is a commerciallyavailable, biphasic, whole blood assay using anenzyme-linked immunosorbent assay (ELISA) tomeasure gamma interferon (IFN c ) production. Theassay was srcinally developed for use in cattle in thediagnosis of tuberculosis, 1 and, with a modification tothe antigen used in the whole blood stimulation phaseof the assay, has subsequently been used in thediagnosis of bovine Johne’s disease. 2 The assaydetects the cell-mediated immune responses that, indiseases such as Johne’s disease, typically occurearlier than the humoral responses that are oftenassociated with onset of clinical disease. The optimalconditions for use of the Bovigam sandwich assay incattle have been well documented. 3 It is recommendedthat bovine blood samples be collected in blood tubesin which heparin salts are used as the anticoagulantand that 16 hr is the optimum incubation time for thewhole blood stimulation phase of the assay. 3 It is alsorecommended that the blood is stimulated within 8 hrof blood collection to obtain maximum IFN c synthesis. 3,4 This is a situation that may be difficultto achieve, particularly with samples collected fromfarm animals in remote field settings.Ovine IFN c  is also detected by the Bovigamsandwich assay 1,5 and, in recent years, the assay hasbeen evaluated as a diagnostic test for use in ovineJohne’s disease (OJD) control programs. 6,7 Theassay’s capacity to detect infected animals early inthe disease process enables identification of poten-tially infected sheep before any bacterial fecalshedding. Irrespective of whether these animalsprogress to the clinical stage, removal of such animalsfrom the flock has the potential to decrease contam-ination of pasture and, subsequently, to reduceinfection of other sheep. However, because of nonspecific IFN c  responses, raised cutoff points havebeen required to achieve high specificity ( $ 98 % ), 7 which has resulted in a reduction of sensitivity to lessthan 50 % , limiting its application for early detectionof infection or certification of disease freedom.Optimization of the IFN c  production in ovine blood From the Farm Animal and Veterinary Public Health, Facultyof Veterinary Science, The University of Sydney, Camden, NewSouth Wales, Australia. 1 Corresponding Author: Katrina L. Bosward, Faculty of Veterinary Science, The University of Sydney, J. L. ShuteBuilding, 425 Werombi Road, Camden, NSW 2570, Vet Diagn Invest 22:210–217 (2010)210  samples may improve the diagnostic sensitivity of theassay. The current study, therefore, aimed to improvethe utility of the Bovigam IFN c  assay a in sheep byinvestigating the effect of anticoagulant, duration of incubation, cell concentration, blood storage temper-ature, time of stimulation of cells with antigen relativeto time of sample collection, and temperature duringtransit on IFN c  production in whole ovine blood. Materials and methods Sheep All experiments using housed or field animals in thepresent study were carried out with the approval of theUniversity of Sydney Animal Ethics Committee. Forthe preliminary investigations, 2 Merino sheep approxi-mately 7 months old were used. Only 2 animals were usedin the study to maximize the number of incubationcondition variables studied. The sheep had been vaccinatedwith an adjuvanted, killed  Mycobacterium avium  subspecies  paratuberculosis  (MAP) vaccine b at least 14 days before theexperiment according to the manufacturer’s instructions.The animals were fed lucerne chaff and grain once dailywith water supplied ad libitum. For the experimentsinvolving naturally infected sheep, Merinos, 3 years of age or older, were obtained from a farm with endemic OJDin the Central Tablelands of New South Wales, Australia,where vaccination is not currently used against the disease.The animals were screened for OJD by agar gel immuno-diffusion (AGID) test. Sixteen AGID-positive sheep weretransported to a containment facility at the University of Sydney, Camden Campus, where the animals were housedand fed lucerne chaff and grain once daily with watersupplied ad libitum. For the large-scale field trial, 50Merino wethers, aged approximately 7 months, wereselected from a property in Trunkey (near Bathurst) inNew South Wales, Australia. These sheep had beenvaccinated b according to the manufacturer’s instructions5 months before blood collection. The choice as to whethervaccinated or naturally infected sheep were used for each of the experiments depended upon availability at the time of the experiment. MAP antigen and blood collection Mechanically disrupted MAP antigen was derived fromstrain 316V. c Blood was collected from the jugular vein intocommercially available 10-ml blood collection tubes, d con-taining 144 United States Pharmacopeia (USP) units of spray-dried lithium–heparin for all experiments, except forthose in which the effect of other anticoagulants wasanalyzed. In these experiments, blood was collected intocommercially available, 10-ml, blood collection tubes d containing either potassium oxalate/sodium fluoride (100/sp, 1,000/ca) or potassium ethylenediamine tetra-acetic acid(EDTA;15 % solution,0.117ml,17.55mg[100/sp,1,000/ca]). Whole blood culture Previous studies in cattle described the nonspecificrelease of IFN c  in some samples when whole blood wasdiluted in tissue culture media. 3 This complication was notobserved with ovine blood samples in the current study, soblood was diluted 1:2 in media to facilitate adequate mixingwith antigen or mitogen. The culture medium used for allexperiments consisted of Roswell Park Memorial Institute(RPMI)-1640 medium, e containing 5 %  fetal bovine serum(FBS), f  L -glutamine f  (2 mM), penicillin–streptomycin e (100  m g/ml), and 2-mercaptoethanol f  (58  m M). For thepreliminary investigations to establish the protocol, 0.5 mlof whole blood was incubated at 37 u C in a humidifiedatmosphere of 5 % CO 2  in air in 48-well culture trays g witheither 0.5 ml RPMI-1640 medium alone or RPMI-164 0medium containing MAP antigen (at a final concentrationof 5  m g/ml). Two mitogens, concanavalin A (Con A) f  at afinal concentration of 10  m g/ml, or pokeweed mitogen(PWM) f  at a final concentration of 5  m g/ml, were used aspositive controls to ensure the cells were capable of responding to stimulation. The concentrations of MAPantigen, Con A, and PWM were optimized by titrationassays, and plates were incubated for up to 72 hr beforeharvesting the supernatant fluid. For all subsequentexperiments, plates were incubated for 48 hr before thesupernatant fluid was aspirated and stored in 96-wellplastic storage plates g at 2 20 u C before being assayed in theBovigam sandwich ELISA. Gamma interferon enzyme-linked immunosorbent assay The whole blood culture method was followed by theBovigam sandwich ELISA as previously described 1 andperformed according to the manufacturer’s instructions.All samples were assayed in duplicate or triplicate, andoptical densities (ODs) were measured on an ELISA platereader h at 450 nm. Samples were reassayed if duplicate ortriplicate values differed from their means by more than10 % . Effect of concentration of peripheral blood leukocytes andtotal and differential peripheral blood leukocyte counts onIFN c  production Concentration of peripheral blood leukocytes.  For theexperiments investigating the effect of concentratingleukocytes (i.e., doubling the total leukocyte count inwhole blood) on the production of IFN c , 10 ml of bloodwas collected into each of 2 commercially available bloodcollection tubes d from 4 naturally infected Merino sheep.One tube was spun in a centrifuge at 1,455 3  g   for 15 min.The buffy coat was then aspirated from this tube, added tothe second blood tube, and mixed thoroughly on a bloodmixer for 10 min before commencing the assay as describedabove. Total and differential leukocyte counts.  Total anddifferential white blood cell counts were performedmanually on blood collected from 16 naturally infectedMerino sheep. Total white blood cell counts wereperformed on cells suspended in Tu¨rk solution usingstandardized methodology, whereas differential cell countswere performed on blood smears stained with a commer-cially available Romanowsky stain i according to themanufacturer’s instructions. Conditions affecting gamma interferon production in sheep 211  Field trial investigations into the effect of varying transporttemperature and time of adding antigen on IFN c  production Blood collection, transport conditions, whole blood assay,and ELISA.  Experiments were conducted on bloodobtained from 50 vaccinated Merino sheep aged approx-imately 7 months and selected from a property in Trunkey(near Bathurst) in New South Wales, Australia. Bloodwas initially collected into commercially available 10-mlblood tubes d containing lithium heparin anticoagulant,and then mixed and immediately dispensed in 1-mlaliquots into 1 of 9 polypropylene-capped tubes. g Threeof the tubes were pretreated with 1 ml of media alone, 3tubes were pretreated with 1 ml of media containing MAPantigen, and 3 tubes were pretreated with 1 ml of mediacontaining PWM. The tubes were then stored at 4 u Covernight and transported to the property at ambienttemperature. The triplicate tubes were then treated in 1 of 3 ways:Group 1.—For each sheep, 3 polypropylene-cappedtubes containing 1 ml of blood in 1 ml of media alone,1 ml of media plus antigen, and 1 ml of media plus mitogen,respectively, were transported to the laboratory at ambienttemperature, which was estimated to vary between 10 u Cand 20 u C. Once at the laboratory, the blood was placed ona laboratory bench overnight at approximately 20 u C, thenincubated at 37 u C for 48 hr before harvesting thesupernatant.Group 2.—For each sheep, 3 polypropylene-cappedtubes containing 1 ml of blood in 1 ml of media alone,1 ml of media plus antigen, and 1 ml of media plus mitogenwere transported to the laboratory in a portable incubator  j set at 37 u C. Once at the laboratory, the blood wasincubated at 37 u C overnight and for a further 48 hr beforeharvesting the supernatant.Group 3.—For each sheep, 3 polypropylene-cappedtubes containing 1 ml of blood in 1 ml of media weretransported to the laboratory at ambient temperature,which was estimated to vary between 10 u C and 20 u C. Onceat the laboratory, the blood was placed on a laboratorybench overnight at approximately 20 u C. The followingmorning, either MAP antigen or PWM was added to 2 of the 3 tubes, whereas the third tube remained as a controltube without additives. All tubes were then placed in thelaboratory incubator at 37 u C for 48 hr before harvestingthe supernatant. Once harvested, all supernatants from all 3groups were stored at  2 20 u C before being assayed in theBovigam sandwich ELISA according to manufacturer’sinstructions. Statistical analyses All statistical analyses were performed using GenStatversion 10, k Minitab version 14, l or SAS release 9.1. m Effect of concentration of peripheral blood leukocytes onIFN  c  production.  Effect of concentration of peripheralblood leukocytes on IFN c  response in blood samplesobtained from 4 naturally infected Merino sheep wasevaluated using a paired  t -test. Effect of total and differential peripheral blood leukocytecounts on IFN  c  production.  Linear regression analyseswere performed to evaluate the effect of total anddifferential leukocyte counts on IFN c  production, mea-sured by OD values of samples containing MAP antigenand by adjusted OD values (OD value MAP  2  OD valuemedia). These outcome variables based on OD values wereregressed on explanatory variables representing the bloodcell counts or percentages, namely white blood cell count,lymphocyte percentage, total lymphocytes, neutrophilpercentage, total neutrophils, monocyte percentage, totalmonocytes, eosinophil percentage, and total eosinophils.All OD values were logarithmically transformed beforeanalyses to normalize their distributions.In addition, logistic regression analyses were performedto evaluate the effect of blood cell counts or percentages onprobability of classification of a sample as positive.Samples were designated as positive or negative using 2stringency criteria. For the more stringent level, the samplewas considered positive if the absorbance of the stimulatedsample (either PWM or MAP antigen) was 0.100 absor-bance units greater than the absorbance achieved for thenonstimulated control well (media only) for that animaland if the absorbance of the stimulated sample was twicethat of the unstimulated sample (i.e., OD MAP/OD mediaonly  $ 2 and OD MAP  $  OD media only  + 0.1). Thisclassification criterion was reported by researchers usingthe Bovigam sandwich assay for detection of IFN c  incattle. 8 For the less-stringent criterion, the sample wasconsidered positive if the absorbance of the stimulatedsample (either PWM or MAP antigen) was at least 0.05absorbance units greater than the absorbance achieved forthe nonstimulated control well (media only) for that animaland if the absorbance of the stimulated sample was at least1.3 times that of the unstimulated sample (i.e., OD MAP/OD media only  $ 1.3 and OD MAP  $  OD media only + 0.05). This criterion has also been reported for thedetection of IFN c  in sheep. 9,10 In initial linear and logistic regression analyses, allexplanatory variables were tested individually to determinetheir unconditional association with the outcome. Subse-quently, explanatory variables associated with the outcomeat univariable  P  -value of  # 0.25 were selected for inclusionin multivariable logistic regression models. Multivariablemodels were fitted using the manual, forward, stepwiseapproach employing the SAS LOGISTIC procedure,retaining variables with  P  -value  , 0.05. Effect of varying transport temperature and time of adding antigen on IFN  c  production.  Optical density values forsamples containing media alone, MAP antigen, and PWMwere compared between 3 treatment groups using analysisof variance. In addition, adjusted OD values, calculated bysubtracting OD values of the samples containing mediaalone from the samples containing MAP antigen, werecompared between 3 treatments. All OD values werelogarithmically transformed before analysis of groupdifferences to normalize their distributions. In a secondset of analyses, OD values were categorized into positiveand negative, and probability of classification of samples aspositive was compared between 3 treatment groups usinglogistic regression analyses. 212  Bosward et al.  Results Effect of anticoagulant The effect of anticoagulant on IFN c  productionwas evaluated in 2 vaccinated animals. Bloodcollected into tubes containing lithium heparinresulted in the optimal response with collection of blood into tubes containing potassium EDTA, andpotassium oxalate/sodium fluoride, resulting in acomplete abrogation of the IFN c  response (Fig. 1). Effect of varying duration of incubation The effect of varying duration of incubation onIFN c  production was evaluated in 2 vaccinatedMerino sheep following stimulation in the wholeblood assay with MAP antigen (final concentration of 5  m g/ml) and Con A mitogen (final concentration of 10  m g/ml). Blood was incubated for 24, 48, or 72 hrbefore harvesting the supernatant. In both sheep andwith both MAP antigen and mitogen, optimum IFN c production was obtained following 48 hr incubation(Fig. 2). The responses to MAP antigen were greaterthan those to ConA in both sheep. Effect of blood storage temperature and duration To account for field sampling and time to assay,the effect on IFN c  production of varying thetemperature at which blood was stored for 4, 8, or24 hr before incubation in the whole blood assay wasevaluated in 2 vaccinated animals. Adding the MAPantigen at the time of blood collection consistentlyresulted in lower IFN c  production than the levelsobtained when the antigen was added just beforeincubation irrespective of the time or temperature atwhich the blood was held before incubation (Table 1).For both sheep, regardless of when antigen wasadded, there were clear indications that the longer thedelay to incubation, the less IFN c  was produced. Effect of total white blood cell concentration and differentialcell count Effect of cell concentration.  Decreasing cell con-centration in vaccinated sheep.—Decreasing theresponding leukocyte population by diluting the totalperipheral blood leukocyte concentration in mediumwas associated with a decreasing IFN c  response(Table 2). There was a substantial reduction in IFN c signal with 2–4-fold reductions in total white bloodcell counts.Increasing and decreasing cell concentration innaturally infected sheep.—The effect of both concen-trating and titration of the peripheral blood leukocyteconcentration with media was examined in 4 naturallyinfected Merino sheep. In all 4 sheep, concentratingthe peripheral blood concentration 2-fold resulted ina significant increase in IFN c  production ( P  -value 5 0.03). Subsequent serial doubling dilutions of thesample in media resulted in a dose-dependentdecreasing IFN c  response (Fig. 3). Effect of total and differential leukocyte counts onIFN  c  production.  These experiments were under-taken on blood obtained from naturally infectedMerino sheep aged  $ 3 years. Linear regressionanalyses were conducted to evaluate the effect of bloodcounts on IFN c  production, but none of the leukocytevariables was significantly associated with MAP ODvalues or MAP-adjusted OD values, indicating thatthey might not influence IFN c  production. Similarly,logistic regression analyses conducted for the 2outcome variables created based on the stringent andthe less-stringent criterion, respectively, suggested noeffect ofbloodcellcounts, exceptthat there were higherodds of a positive outcome (stringent) in animals withhigh total lymphocyte counts. Figure 1.  Effect of anticoagulant on gamma interferonproduction during incubation of whole blood with  Mycobacteriumavium  subspecies  paratuberculosis  antigen (at a final concentrationof 5  m g/ml) and measured as optical density (OD) at 450 nmin the Bovigam sandwich assay in 2 vaccinated Merino sheep.EDTA  5 ethylenediamine tetra-acetic acid. Figure 2.  Effect of varying whole blood incubation time at37 u C on gamma interferon production in peripheral bloodobtained from 2 vaccinated Merino sheep in response tostimulation with  Mycobacterium avium  subspecies  paratuberculosis (MAP) antigen (final concentration of 5  m g/ml) and concanavalinA (Con A) mitogen (final concentration of 10  m g/ml) andmeasured as optical density (OD) at 450 nm in the Bovigamsandwich assay.Conditions affecting gamma interferon production in sheep 213
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