Multiplex screening of surface proteins from Mycoplasma ... · 3 1 INTRODUCTION 2 3 Contagious...

1

Multiplex screening of surface proteins from Mycoplasma mycoides 1

subsp. mycoides SC for an antigen cocktail ELISA 2

Maja Neiman1, Carl Hamsten

1, Jochen M. Schwenk

1, Göran Bölske

2 and Anja Persson

1* 3

4

1Department of Proteomics, School of Biotechnology, KTH - Royal Institute of 5

Technology, AlbaNova University Center, SE-106 91 Stockholm, Sweden 6

2Department of Bacteriology, National Veterinary Institute, SE-75183 Uppsala, 7

Sweden 8

* Corresponding author. Phone: +46-8-5537 8017 Fax: +46-8-5537 8481. E-mail: 9

[email protected]. 10

11

Running title: Antigen cocktail ELISA 12

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Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00223-09 CVI Accepts, published online ahead of print on 2 September 2009

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ABSTRACT 1

2

A recombinant antigen cocktail ELISA for diagnosis of contagious bovine 3

pleuropneumonia (CBPP) was developed after careful selection of antigens among 4

one third of the surface proteome of the infectious agent Mycoplasma mycoides subsp. 5

mycoides SC (M. mycoides SC). First, a miniaturized and parallelized assay system 6

employing antigen suspension bead array technology was used to screen 97 bovine 7

sera for their humoral immune response towards 61 recombinant surface proteins 8

from M. mycoides SC. Statistical analysis of the data resulted in selection of eight 9

proteins that showed strong serologic responses in CBPP-affected sera and minimal 10

reactivity in negative control sera, with p-values less than 10-6

. Only minor cross 11

reactivity to hyperimmune sera against other mycoplasmas was observed. When 12

applied in an ELISA, the cocktail of eight recombinant antigens allowed a five fold 13

signal separation between 24 CBPP-affected and 23 CBPP-free sera from different 14

geographical origin. No false positives and only two false negatives were obtained. In 15

conclusion, the selected recombinant mycoplasma antigens qualified as highly 16

specific markers for CBPP and could be employed in both a suspension bead array 17

platform and a cocktail ELISA setting. This set of proteins and technologies therefore 18

offer a powerful combination to drive and further improve serological assays towards 19

reliable, simple and cost-effective diagnosis of CBPP. 20

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INTRODUCTION 1

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Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease in cattle 3

caused by Mycoplasma mycoides subspecies mycoides small colony type (M. 4

mycoides SC). Because of its potential for rapid spread with resulting massive losses 5

of livestock and thereby severe socioeconomic consequences, an official declaration 6

to the World Organisation for Animal Health (OIE) is required. Vigorous and costly 7

eradication programs involving mass slaughter, quarantine and strict control of animal 8

movements have been successful in eradicating CBPP from USA, Japan and Australia 9

(13). In Western Europe, the disease has reemerged almost every decade in the 20th

10

century in spite of expensive eradication efforts, demonstrating its constant threat (13, 11

22). In Sub-Saharan Africa where the disease is endemic, this stamping out procedure 12

is not economically feasible. Vaccination is better option for these countries, although 13

existing vaccines so far give insufficient immunity and severe side effects (23). One 14

of the main challenges within CBPP control is diagnosis of the subacute and chronic 15

phases of the disease (14, 22). Without proper diagnosis, asymptomatic carriers can 16

easily transmit the pathogen and incubation periods up to several months (7) hinder 17

contact tracing. Today, there are two diagnostic tests prescribed for international 18

trade; the Campbell and Turner complement fixation test (CFT) established in 1953 19

(6) based on whole cell antigens and the competitive ELISA (cELISA) published in 20

1998 (14) based on whole cell antigens in combination with a monoclonal antibody 21

towards Pts-G (9). Although the two serological tests supplement each other in 22

sensitivity, they still do not allow adequate diagnostic certainty (8). 23

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The complete genome sequence of the M. mycoides SC (26) has paved the way for 1

new diagnostics based on subcellular components. Methods including PCR have been 2

shown successful (21), but put high demands on sampling procedures. Surface located 3

lipoproteins are of high interest both for diagnostic purposes as well as for studies 4

regarding the pathogenicity of the bacteria (23). Up to date only a few of the surface 5

located lipoproteins from M. mycoides SC have been studied thoroughly. LppA (p72) 6

(17, 18), LppB (25) and LppC (23) are highly conserved lipoproteins that are present 7

in closely related species within the Mycoplasma mycoides cluster. Pts-G is a variably 8

expressed glucose phosphotransferase system permease (9) and Vmm is a small 9

surface protein shown to have a variable expression pattern (20). LppQ is a highly 10

antigenic lipoprotein specific to M. mycoides SC (1). Thorough characterization 11

studies of LppQ (1, 4) and the development of a recombinant ELISA built upon LppQ 12

as antigen (5) show that it is suitable as a diagnostic marker. However, out of the 187 13

predicted surface proteins of M. mycoides SC (10), more antigens than just LppQ 14

should trigger antibody mediated immune responses useful in diagnostic applications. 15

Combinations of such antigens could thereby offer a higher specificity and sensitivity 16

than existing methods by adding discriminative power to current LppQ-based ELISA 17

while circumventing cross reactivity compared to whole cell antigen based methods. 18

19

The aim of this study was to identify the most potent diagnostic surface antigens and 20

to test the performance of recombinant versions in combination in an ELISA format. 21

The selection of targets was enabled by a recently developed multiplex suspension 22

bead array assay that allowed high-throughput screening of humoral immune 23

responses in a large set of sera, against a large number of recombinant surface 24

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proteins (10). As a result, a cocktail ELISA was developed with the selected antigens 1

and its capacity was evaluated. 2

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MATERIALS AND METHODS 4

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Recombinant surface proteins. All proteins included in this study are listed in 6

supplemental table S1. The production of recombinant mycoplasma proteins was 7

described previously (11), and included the selection of surface proteins specific to M. 8

mycoides SC, design of full length recombinants excluding signal peptide and 9

including the largest extracellular domain for transmembrane proteins, PCR and 10

cloning into the vector pAff8c, mutagenesis to exchange TGA codons to TGG, 11

expression in E. coli and finally purification by immobilized metal ion 12

chromatography. All recombinant proteins were equipped with a fusion tag consisting 13

of a 17 kDa albumin binding protein with six N-terminal histidines (His6ABP) for 14

enhanced solubility and purification purposes. 15

16

For the ELISA set-up, relevant recombinant proteins were also produced without the 17

fusion protein ABP. New forward PCR primers were designed to replace the N-18

terminal His6ABP fusion tag with a hexahistidine tag only. Sequence-specific regions 19

were identical to previously used PCR primers but the primer handles contained a 5’-20

terminal NcoI restriction site followed by a hexahistidine tag. A biotinylated primer 21

complementary to this handle was used in a secondary PCR step to enable automated 22

solid phase restriction of the amplified mycoplasma gene fragments. Amplifications 23

were performed as previously described, using the sequence verified clones with 24

His6ABP gene fusions as template. After solid phase restriction, clones were ligated 25

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into NcoI/AscI digested pAff8c vector. Proteins were expressed in E. coli strain 1

BL21(DE3) and purified using immobilized metal ion affinity chromatography. All 2

proteins were stored at -20°C in phosphate buffered saline (PBS) containing 1 M Urea 3

and were named after their corresponding open reading frame in the genome 4

sequence. 5

6

Serum collection. The serum collection consisted of 97 bovine sera from different 7

geographical origins as well as 17 hyperimmune sera (Table 1). Included in the 8

collection were two CBPP-positive reference samples from Portugal, 50 CBPP-9

positive field samples from Spain, Namibia and Tanzania, 24 CBPP-negative field 10

samples from Kenya and Tanzania, 21 negative control samples from Sweden and 17 11

hyperimmune sera from Denmark and Sweden. The bovine sera had well-12

characterized CBPP-status, although some diagnostic test results were inconsistent. 13

The hyperimmune sera were from four calves and 13 rabbits immunized with M. 14

mycoides SC type strain PG1 as well as closely related Mycoplasma species to study 15

potential cross reactivity. 16

17

Screening by the suspension bead array assay. The assay was performed as 18

previously described (10) on a total of 134 samples with a 62-plex surface protein 19

array in suspension. The array consisted of sixty-one recombinant mycoplasma 20

proteins and pure His6ABP covalently coupled to colour coded magnetic 21

microspheres (MagPlex, Luminex Corp.). Coupling efficiency was confirmed and 22

bead collections were combined and optimized to a final concentration of 100 beads 23

per ID and µl. Sera were diluted 1:3000 and unspecific binding was reduced by pre-24

adsorption with His6ABP protein and E. coli lysate. Incubation of sera and protein-25

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coupled beads was followed by detection of bound complex by a biotinylated 1

secondary antibody and fluorophor labeled streptavidin. Fluorescence intensity was 2

registered in a Lx200 system using xPONENT software (both Luminex Corp.). 3

Signals were chosen from the median fluorescence intensity (MFI) of at least 50 4

beads per bead-ID and are given in arbitrary units (AU). The obtained results were 5

validated according to bead count, technical replicates and background levels. 6

Statistical analysis for the selection of the most immunogenic antigens as well as 7

visualization of the data was performed with R, a web based statistical computing 8

language and environment (12). A heat map visualisation was based on log10-9

transformed intensity values and cluster analysis was performed with hierachical 10

clustering based on the Euclidean distance between data points. Subsequent selection 11

of antigens was based on Welch’s two sample t-test on log-transformed data where 12

antigens with a positive to negative signal ratio of at least 3 and a p-value below 0.01 13

were selected for further evaluations. 14

15

Antigen cocktail ELISA. A cocktail of the most immunogenic antigens was created 16

by combining selected recombinant proteins in equal amounts in PBS. ELISA 17

conditions were optimized by varying protein coating density (10-1000 ng/well), plate 18

type (high binding or medium binding half well area 96-well plates, Greiner), reagent 19

volumes (50-100 µl), serum dilutions (1:10-1:1600), secondary antibody 20

concentrations (80-160 ng/ml), incubation times (10-60 min) and washing procedures 21

(rounds, volumes and addition of Tween20 to PBS). The final assay was conducted as 22

follows: ELISA microplates (high binding half area plates, Greiner) were coated with 23

protein cocktail (100 µl, 1 µg/ml) and incubated overnight at 4 ºC. After washing 24

three times in 140 µl PBS, plates were blocked with buffer BRE (140 µl, Blocking 25

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Reagent for ELISA, Roche) at RT for 1 h, followed by three washes of 140 µl PBST 1

(PBS with 0.1 % Tween 20). Sera were diluted 1:1000 in BRE, and 100 µl were 2

dispensed into each well and incubated for 1 h at RT. After three washes, 100 µl anti-3

bovine IgG conjugated to horseradish peroxidase (80 ng/ml, Jackson 4

Immunoresearch) were added and incubated for 1 h at RT. After washing the 5

enzymatic color reaction was performed by the addition of 100 µl 1-step™ ABTS 6

(2,2'-azinobis [3-ethylbenzthiazoline-6-sulfonic acid] diammonium salt, Pierce) and 7

the optical density (OD) was measured at 405 nm (SUNRISE absorbance reader, 8

Magellan software, TECAN) after 30-60 min. 9

10

RESULTS 11

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Recombinant production of surface proteins. Sixty-one proteins with predicted 13

surface location on M. mycoides SC were produced as recombinant proteins with the 14

fusion tag His6ABP. Protein yields from 100 ml culture after affinity purification 15

ranged from 0.2 to 10 mg. For nine selected antigens, ABP-free recombinants were 16

produced for the final ELISA. Protein yields were similar to those having the ABP 17

fusion and ranged from 0.6 to 7.3 mg. However, for recombinant R0816, expression 18

of a tag-free version failed and it was therefore excluded. 19

20

Screening of sera against 61 recombinant proteins. In order to select the most 21

immunogenic among 61 surface proteins from M. mycoides SC, a screening of sera to 22

monitor the humoral immune response was performed. By a suspension bead array 23

assay, a multiplex and simultaneous analysis of immunoglobulins with reactivity to 24

the recombinant surface proteins was achieved for every serum sample. The signal 25

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intensities from all recombinant proteins formed specific and individual patterns that 1

were shown to be reproducible for each serum in our previous work (10). Signal 2

intensities in technical replicates varied with an average intra assay coefficient of 3

variation (CV) of 21%. The serum free control, used to monitor unspecific binding of 4

secondary antibodies to the protein-coated beads, displayed signals that peaked at 3-5

20 AU, the intensities equivalent to the intrinsic auto-flourescence of the beads. The 6

control bead carrying the tag protein His6ABP displayed a median MFI of 45 AU, 7

reflecting binding of serum antibodies to the fusion partner of the proteins. To 8

distinguish between noise and antibody-mycoplasma antigen signal, an MFI threshold 9

of 142 AU was set. This threshold was based on the 95th

percentile of the distribution 10

of signal intensities from the His6ABP-bead. Intensity levels exceeding this threshold 11

value were judged as specific binding of serum antibodies to the recombinant 12

mycoplasma proteins. 13

14

Distinct differences between CBPP-affected and CBPP-free sera were seen in the 15

serum patterns. For 19 proteins the signal median in CBPP positive sera was 16

significantly above the threshold value of 142 AU when performing a Wilcoxon rank 17

sum test, as compared to two proteins in the CBPP negative sera. For the majority of 18

the proteins however, signals were less than the threshold value of 142 AU. Sera 19

categorized as CBPP positive showed a higher overall average intensity (484 AU) 20

compared to CBPP-negative sera (106 AU). Swedish negative control sera were found 21

to yield even lower overall average signal intensity (64 AU) than the CBPP-free 22

African field sera (151 AU). 23

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The screening data were summarized in a heat map in combination with a cluster 1

analysis on both serum and protein level (Figure1). The sera separated into five main 2

clusters, denoted I-V. All technical replicate samples clustered together, except for 3

one specimen of sera B079. Clusters I, II and V consisted of 25, 20 and 16 samples 4

respectively, whereof 25, 13 and 16 samples were from CBPP-positive sera. These 5

clusters contained most of the high signal intensity data points. All hyperimmune 6

sera, except for the M. mycoides SC PG1 antiserum, clustered together in cluster III 7

that displayed particularly low signals, together with five sera from Swedish bovines 8

infected with Mycoplasma bovis (B094-B098), one Swedish negative control (B084) 9

and one CBPP-positive sample (B028). Cluster IV consisted of 50 samples whereof 10

45 were CBPP-negative (including ten replicates of a negative control pool, B093), 11

four were CBPP-positive and one was the M. mycoides SC PG1 antiserum (H099). In 12

the vertical protein dendrogram, the top cluster consisting of nine proteins appeared 13

with potentially diagnostic importance. High amounts of antibodies were detected to 14

these proteins in CBPP-positive sera. One of them, R1046 (LppQ), yielded 15

particularly high signals in CBPP-positive sera. 16

17

Selection of antigens. Selection of the most immunogenic antigens among the 61 18

recombinant proteins was based on Welch’s two sample t-test. This test was 19

performed on a log-transformed data set where blanks, replicates, hyperimmune sera 20

and Swedish control sera were removed. Thus, the statistical evaluation involved 24 21

CBPP-negative field samples, 50 CBPP-positive field samples and two positive 22

reference samples. The test revealed that among the 61 recombinant mycoplasma 23

surface proteins, eight proteins had statistically significant positive to negative signal 24

ratios greater than 3 with p-values ranging from 10-6

to 10-18

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proteins were R1046, R0136, R0653, R0431, R0816, R0813, R0240 and R0397, all 1

belonging to the protein cluster of appearent diagnostic importance (Figure 1). As the 2

theoretically most discriminative and therefore most potent diagnostic antigens among 3

the investigated surface proteins, these eight were selected as candidates for further 4

evaluation (Table 2). Additionally four recombinant proteins, R0051, R0711, R0570 5

and R0322, appeared with p-values below the significance threshold 0.01 but their 6

discriminating power, reflected by both the p-value as well as the fold change 7

difference, was not as good as the above eight. An additional cluster analysis was 8

performed on the screening data employing all bovine sera with only the eight 9

candidate antigens and a distinct separation of samples into one CBPP-positive and 10

one CBPP-negative cluster was observed (Figure 3). Five of the seven CBPP-negative 11

sera that previously grouped with positives in cluster II in Figure 1 now cluster with 12

the CBPP-negative group, but there are also three additional positives in the negative 13

cluster. However, the cluster analysis is by no means a diagnostic test, but a 14

visualisation of signal similarities between samples. By presenting the distribution of 15

signal intensities in the positive and negative populations in a box plot (Figure 4), 16

each antigen’s discrepancy between CBPP-positive samples and CBPP-negative 17

samples is shown. None of the eight proteins was discriminative enough to fully 18

distinguish the positives from the negatives alone, thus supporting that an antigen 19

cocktail could offer an improved alternative for building a diagnostic system. 20

21

From the associated study (10) three additional mycoplasma surface proteins became 22

available at a later stage and increased the number of mycoplasma proteins 23

investigated to 64. One of these antigens, denoted R0079, displayed its potential as a 24

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CBPP-marker (data not shown) and was therefore included into the following 1

evaluation and ELISA development. 2

3

Among the proteins corresponding to the nine selected recombinants, only LppQ 4

corresponding to R1046 have previously been described as immunodominant. The 5

protein corresponding to R0816 is annotated as a putative variable surface protein, 6

and has been used in our previous work to study immune responses (10, 11). The 7

protein corresponding to R0397 is a putative lipoprotein whose gene was indentified 8

in a screening to find candidate genes for a DNA vaccine against CBPP (15), and a 9

protective effect was shown for the protein in a mycoplasmaemia mouse model. There 10

is no information published on the remaining six proteins except the bioinformatic 11

annotation of the genomic sequence. The protein corresponding to R0813 is annotated 12

as a putative variable surface protein and show resemblance to R0816, as a ClustalW 13

alignment gave a pairwise score of 48. Three of the proteins, corresponding to 14

recombinants R0240, R0431 and R0653, are putative lipoproteins while the remaining 15

two, corresponding to R0079 and R0136, are annotated as putative phosphonate ABC 16

transporter phnD and putative protein respectively. The gene MSC_0136 17

corresponding to the recombinant R0136, has previously been used in a phylogenetic 18

study of the Mycoplasma mycoides cluster (24). The Interpro motif IPR005046 found 19

in predicted surface proteins of many bacteria is shared by six proteins, corresponding 20

to R0136, R0431, R0653, R0813, R0816 and R1046. Sizes of the selected 21

recombinant proteins, excluding His6ABP, range from 20 to 60 kDa and their native 22

counterparts range from 26 to 64 kDa. A summary of the selected antigens is 23

presented in Table 2. 24

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Cross reactivity. In the process of evaluating the recombinant proteins as antigens of 1

a diagnostic system, the cross reactivity to closely related Mycoplasma species was 2

investigated. Hyperimmune sera from rabbits and calves immunized with closely 3

related species were included in the study and their immune responses towards all 61 4

proteins were analyzed. In the suspension bead array assay these samples showed 5

serum patterns distinctly different compared to the bovine sera. Patterns consisted of 6

signals of 10-20 AU for almost all proteins, within the range of bead intrinsic auto-7

fluorescence and below the noise level threshold of 142 AU. Only a handful of 8

proteins gave rise to higher signals of 200-5000 AU. Among these samples, the 9

highest signals were noted for the positive control M. mycoides SC type strain PG1 10

antiserum. The hyperimmune sera from calves had the same profiles as the rabbit sera 11

thus excluding that results were caused by the anti-rabbit secondary antibody. Five 12

bovines from Sweden with M. bovis-infections were included (B094-B098) to 13

evaluate cross reactivity to M. bovis antigens and were more comparable to the other 14

bovine sera than the hyperimmune rabbits and calves. These samples displayed no 15

sign of cross reactivity as all signals were below the noise-level of 142 AU. Cross 16

reactivity intensities for the eight selected antigens in the hyperimmune sera are 17

summarized in Table 3. A minor cross reactivity was observed for some antisera from 18

the M. mycoides cluster (H099-H104). 19

20

Effect of ABP in the suspension array assay. A diagnostic system to monitor 21

bacterial infection should preferably not be built on recombinant proteins with 22

components of bacterial origin that could cause false positive results. To verify that 23

our screening could be relevant for tag-free recombinant proteins as well, eight 24

proteins with and without ABP as well as the His6ABP control were tested with 47 25

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sera on the suspension bead array. Hereby it was shown that the separation between 1

CBPP positive and CBPP negative sera was not affected by the presence or absence of 2

ABP (data not shown). Generally, an increase in signal intensity was observed for 3

recombinant proteins without fusion tag, possibly associated with bead coating 4

properties. 5

6

Development of an antigen cocktail ELISA. A protein cocktail of the eight ABP-7

free proteins was used in an ELISA setup and optimization experiments were 8

performed to establish a robust protocol. Antigen coating concentrations and serum 9

dilutions were varied to obtain signal intensities with the largest possible signal fold 10

change between CBPP-positive and CBPP-negative sera. It was found that a protein 11

coating concentration of 100 ng/well and serum dilution 1:1000 allowed the best 12

discriminative power. The surface and quality of the ELISA plate was crucial to 13

obtain a robust assay. The intra assay coefficient of variation was reduced from 17% 14

to 3% by changing the plastic from a particularly hydrophobic medium binding plate, 15

to one spiked with polar groups and referred to as high binding. The most important 16

parameter to achieve high signal intensity levels, and thus large signal fold changes, 17

was the incubation time for developing the colorimetric substrate reaction. With the 18

approach used, a signal separation between positive and negative sera was visible 19

after 10 minutes, but the discrepancy increased with time. After one hour, signals 20

stabilized and this incubation time was therefore selected. The colorimetric signals 21

were stable enough for the same relative results to be determined by re-measuring on 22

the following day. 23

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Performance of the antigen cocktail ELISA. In the final set-up of the eight antigen 1

cocktail ELISA, sera were analyzed in comparison to reference serum B001 (16), and 2

signals were reported in percent positivity (27). Evaluation of assay reproducibility 3

with two experiments of triplicates of 15 sera showed an average intra assay CV of 4

3% and an average inter assay CV of 8% (Figure 5). In a proof-of-concept study with 5

47 sera performed twice (Figure 6), the assay allowed a five-fold signal ratio 6

separation between CBPP-affected sera (105% average) and CBPP-free sera (22% 7

average). Receiver operator characteristics of the assay showed a high diagnostic 8

capability since the area under the curve was calculated to 0.991. An initial cut-off in 9

percent positivity of 50% resulted in no false positives and two false negatives with a 10

percent positivity of 29% (B011) and 31% (B012) respectively. These two sera were 11

both supplied with inconsistent results in serological tests(Table 1). Both B011 and 12

B012 had positive results from latex agglutination test as well as gross pathology. 13

B011 had a positive CFT-titer while B012 had negative CFT-titer and both sera were 14

negative on the competitive ELISA. Two negative sera had a percent positivity of 15

48% and 49% respectively which is close to the cut-off and would prompt an 16

additional analysis in a diagnostic situation. As observed by the bead based array 17

assay, Swedish and African negative sera differed in intensity levels. Swedish sera 18

had an average of 17% and African 24% positivity. It needs to be stated that even 19

though origin–dependent variation in intensity levels was observed, no 20

misclassification could be assigned to this. 21

22

DISCUSSION 23

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In the presented study, a bead-based suspension array assay was applied to discover 1

the most immunogenic surface antigens from M. mycoides SC among 64 2

recombinantly produced surface proteins, corresponding to a third of the bacteria’s 3

predicted surface proteome. Large scale screening of bovine sera resulted in the 4

selection of nine proteins to be antigen candidates for the development of enhanced 5

serological diagnosis of CBPP. Eight of these were combined in an antigen cocktail 6

and used to build an ELISA for diagnosis of M. mycoides SC-infection. 7

8

The bead-based suspension array assay proved to be a powerful tool to monitor a 9

multitude of humoral immune responses. Finger print like immune response patterns 10

from sera of CBPP-affected cattle appeared to reflect the individuality of humoral 11

responses to different mycoplasma antigens and supported the idea of using multiple 12

antigens in diagnostic tests. Importantly, objective numerical data was obtained, why 13

it was possible to apply a statistical significance test to select the antigens with best 14

discriminative power between CBPP-positive and negative populations. The Swedish 15

control samples needed to be excluded because the statistical analyses showed 16

significant differences also between the Swedish negative controls and the African 17

negative controls. We could thereby conclude that in using the suspension bead array 18

assay for selection of proteins that discriminate between sample populations, it is 19

important to minimize differences between the groups and preferably use samples of 20

similar origin and collection procedure. In this case the Swedish sera appeared to have 21

a lower general antibody titer compared to the African field sera, probably due to 22

differences in exposure to infectious agents and other environmental factors. Still, the 23

21 Swedish control sera were important as technical controls to monitor assay induced 24

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artifacts. Noteworthy is that the same top candidate diagnostic proteins were 1

identified whether or not the Swedish samples were included. 2

3

The His6ABP tag of the recombinant proteins, used for purification and to enhance 4

solubility, played an important role in the suspension bead array assay as its presence 5

made it possible to measure coating density of the beads with an anti-ABP antibody. 6

By including a bead carrying the fusion partner His6ABP per se, cross-reactivity to the 7

His6ABP tag was monitored in the suspension bead array assay. An option in the data 8

analysis was to use the signal from this negative control to normalize signals between 9

wells, but no correlation between response towards ABP and specific antibody signals 10

was observed. The negative control signal was instead used to define a threshold 11

value, where intensity levels exceeding this threshold were judged as signals from 12

serum antibodies binding to the recombinant mycoplasma proteins. During 13

optimization of the ELISA though, the fusion protein ABP was excluded since the 14

experimental layout would require to additionally coat wells with His6ABP, as in 15

other antigen ELISA:s based on a fusion protein (2). 16

17

For an adequate diagnosis of CBPP, minimal cross-reactivity to related 18

mycoplasmoses is crucial. Surprisingly, the suspension bead array profiles from the 19

17 hyperimmune sera were found to be different from the African bovine sera, and 20

signals were obtained from antibody recognition of a few recombinant proteins only 21

also in the positive control anti-PG1 serum. However, those proteins that did appear 22

were also among the most immunopotent surface proteins selected from the analysis 23

of CBPP-sera. It therefore seems reasonable that any high signal intensity value is 24

representative for a case of infection but ”artificial” immunization appears to give a 25

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more selective immune response. Therefore, field samples from outbreaks of other 1

mycoplasma infections would offer a preferable cohort in studies like this one. 2

Nonetheless, the native protein corresponding to protein R1046 known as LppQ has 3

previously been shown to cross react with antisera to Mycoplasma mycoides subsp. 4

capri, Mycoplasma capricolum subsp. capripneumoniae, and Mycoplasma sp. bovine 5

group 7 (1) and these were the same species found to cross react to R1046 in this 6

investigation. In a study of antigenic specificity of LppQ (1), it was found that 7

antisera raised against the C-terminal LppQ-C’ showed reactivity to proteins present 8

in the closely related species while no cross-reactivity was detected for the N-terminal 9

part of LppQ. This opens the possibility for more extended future studies that include 10

thorough domain analysis of the selected antigens. Subcloning of the proteins could 11

then offer a valuable strategy to adapt the target antigens to increase the specificity in 12

the presented diagnostic application. 13

14

All serologic diagnostic methods are influenced by alternations in the immune 15

response of infected animals. Antibody titers are highly individual and have been 16

reported to lack relationship to the severity of the lesions (19), and chronically 17

infected animals may additionally not be detectable with serological tests. This is why 18

results from serological tests are most confident on a herd level. It poses a challenge 19

to determine specificity of a new serological test when existing methods give 20

inconsistent results and it is therefore required to link experimental data directly to 21

clinical data. The serum collection available for this study were limited to include sera 22

from bovines that had adequate diagnostic information. As was observed in our 23

analysis, several transport events and storage times might have affected sample 24

quality and this could, among other things, effect the discovery of false negatives. 25

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19

Since none of today’s serologic method is sufficiently sensitive per se, it is difficult to 1

fully evaluate the performance of the antigen cocktail ELISA. A reason for false 2

positives discovered in our analysis could be due to a superior sensitivity of the bead 3

based array assay as compared to traditional serology systems. Results from the well 4

characterised reference sera B001 and B002 were in clear concordance to expected 5

and reported results (16) in both the bead based assay as the cocktail ELISA. 6

7

The final antigen cocktail ELISA of this investigation had a discriminative power that 8

distinguished sera from CBPP-affected and CBPP-free bovines. Thus a promising 9

ELISA setup has successfully evolved from the screening assay. However, to become 10

a solid diagnostic ELISA, future evaluation on larger cohorts of preferably fresh 11

bovine sera from herds of various geographical origin are needed to optimize 12

positivity thresholds in order to determine the sensitivity and specificity. It is 13

preferred to conduct future studies in facilities at the site of livestock affected by 14

CBPP to allow a direct comparison between the cocktail ELISA and other available 15

tests. 16

17

In conclusion, the selected recombinant mycoplasma antigens qualified as highly 18

specific markers for CBPP and could be employed in both a suspension bead array 19

platform as well as in a cocktail ELISA setting. This set of proteins and technologies 20

therefore offer a powerful combination to drive and further improve serological 21

assays towards reliable, simple and cost-effective diagnostics of CBPP. 22

23

ACKNOWLEDGEMENTS 24

25

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For providing sera to this study we wish to thank the late Dr Otto Hübschle (CVL, 1

Namibia), Dr Roger Ayling (VLA, UK) and Dr John March (BigDNA Ltd, UK). We 2

would also like to thank Dr Peter Nilsson and Daniel Klevebring (KTH, Sweden) for 3

support and fruitful discussions on data analysis. This study was funded by the 4

Swedish International Development Cooperation Agency (SIDA). 5

6

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2

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characterization of lipoprotein LppQ from Mycoplasma mycoides subsp. 4

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2008. Characterisation of Mycoplasma capricolum P60 surface lipoprotein and 8

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6. Campbell, A. B., Turner, A.W. 1953. Studies on Contagious bovine 21

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21. 5

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membrane protein gene Veterinary Microbiology 72:251-268. 11

25. Vilei, E. M., E. M. Abdo, J. Nicolet, A. Botelho, R. Goncalves, and J. Frey. 12

2000. Genomic and antigenic differences between the European and 13

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E. Johansson, B. Pettersson, and M. Uhlen. 2004. The genome sequence of 17

Mycoplasma mycoides subsp. mycoides SC type strain PG1T, the causative 18

agent of contagious bovine pleuropneumonia (CBPP). Genome Res 14:221-7. 19

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24

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TABLES AND FIGURES 1

2

Table 1 – Serum collection 3

Serum type

Serum ID1

Diagnosis2 Origin Comment

CFT cElisa LAT Pat

CBPP-positive sera

B001 + na na na Portugal4 CBPP reference serum 845 from OIE reference laboratory for CBPP

(52 sera) B002 + na na na Portugal4 CBPP reference serum 840 from OIE reference laboratory for CBPP

B003-B008

na na na na Spain5 Field samples from outbreaks in Spain

B009 + + + - Namibia Field samples from CBPP-outbreak (2004)

B010 - + + - Namibia Field samples from CBPP-outbreak (2004)

B011 + - + + Namibia Field samples from CBPP-outbreak (2004)

B012 - - + - Namibia Field samples from CBPP-outbreak (2004)

B013 - + + - Namibia Field samples from CBPP-outbreak (2004)

B014 + ? + - Namibia Field samples from CBPP-outbreak (2004)

B015 + + ? + Namibia Field samples from CBPP-outbreak (2004)

B016 + + + + Namibia Field samples from CBPP-outbreak (2004)

B017 + +3 na na Tanzania From acute CBPP case (1990) Diluted 1:4 for use as reference serum

B018-B021

+ +3 na na Tanzania Field samples from CBPP-outbreak (1990)

B022 - -3 na na Tanzania Field samples from CBPP-outbreak (1990)

B023-B027

- +3 na na Tanzania Field samples from CBPP-outbreak (1990)

B028-B052

+ na na na Tanzania Field samples from CBPP-outbreak

CBPP-negative sera

B053-B060

na - - na Kenya CBPP-free members of vaccine trial, before start of trial (1998)

(46 sera) B061-B076

- -3 na na Tanzania From CBPP unaffected region (1990)

B077-B092

na na na na Sweden From CBPP-free region (2007)

B093 na na na na Sweden Pool of B081-B092

B094-B098

na -3 na na Sweden From CBPP-free region, with M. bovis infection (1988)

Hyperimmune sera

H099 na na na na Denmark6 Rabbit immunized with M. mycoides SC PG1

(17 sera) H100 na na na na Sweden Rabbit immunized with M. mycoides LC Y-goat

H101 na na na na Sweden Rabbit immunized with M. mycoides capri PG3

H102 na na na na Sweden Rabbit immunized with M. capricolum CK

H103 na na na na Sweden Rabbit immunized with M. capripneumonie F38

H104 na na na na Sweden Rabbit immunized with M. sp. Gr. 7 PG50

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H105 na na na na Denmark6 Rabbit immunized with M. bovis PG45

H106 na na na na Sweden Rabbit immunized with M. bovirhinis PG43

H107 na na na na Sweden Rabbit immunized with M. arginini G230

H108 na na na na Denmark6 Rabbit immunized with M. californicum ST-6

H109 na na na na Denmark6 Rabbit immunized with M. bovoculi M165/69

H110 na na na na Denmark6 Rabbit immunized with M. canadense 275c

H111 na na na na Sweden Rabbit immunized with M. bovigenitalium PG11

H112 na na na na Sweden Calf immunized with A. laidlawii

H113 na na na na Sweden Calf immunized with M. arginini

H114 na na na na Sweden Calf immunized with M. fermentans

H115 na na na na Sweden Calf immunized with M. orale

1 B = bovine sera, H = hyperimmune sera 1

2 CFT = Complement Fixation Test, cELISA = Competivite ELISA, LAT = Latex Agglutination Test, Pat 2

= Gross Pathology, + = positive test result, - = negative test result, ? = incunclusive test result, na = 3

not available 4

3 Indirect ELISA (3) 5

4 OIE Reference Laboratory for CBPP, Lisboa, Portugal (Dr. J. Regalla) 6

5 Laboratorio de Sanidad Animal, Santa Fe, Granada, Spain (Dr. F. Garrido Abellan) 7

6 WHO/FAO Collaborating Centre for Animal Mycoplasmas, University of Aarhus, Aarhus, Denmark 8

(Prof E.A. Freundt) 9

10

Table 2 – Selected recombinant mycoplasma antigens 11

Recombinant

protein

Native

protein1

P-value from

suspension bead

array screening

Included in

cocktail

ELISA

Recombinant

size (kDa)

Native size

(kDa)

R1046 LppQ 5.4 e-18 Yes 49.7 52.1

R0136 Put protein 2.5 e-15 Yes 32.7 37.2

R0431 Put lipoprot 1.5 e-9 Yes 38.4 40.8


R0813 Put vsp 3.4 e-8 Yes 58.6 57.9

R0816 Put vsp 1.2 e-7 No 45.1 46.0



R0079 ABC-

transporter

Not included in

screening

Yes 49.3 50.1

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1 Put lipoprot = putative lipoprotein, Put vsp = putative variable surface protein 1

2

Table 3 – Cross reactivity. Screening results with hyperimmune sera for different 3

mycoplasma species (H099-H115) to detect reactivity to the selected eight 4

recombinant proteins and the His6ABP control. Values are given as MFI (AU). Bold 5

values indicates intensities greater than the noise threshold of 142 AU, thus predicting 6

cross reactivity in a diagnostic set-up. Some cross reactivity was detected for samples 7

within the M. mycoides cluster (H099-H104). 8

Serum

ID

R1046 R0136 R0653 R0431 R0816 R0813 R0240 R0397 His6ABP

H099 5827 16 14 20 14 22 285 2074 20

H100 58 14 25 16 31 26 39 20 16

H101 2601 77 30 36 41 172 280 1523 31

H102 47 15 13 8 11 46 18 514 16

H103 1184 18 19 19 17 28 1465 119 18

H104 2812 23 24 1510 19 18 7 1446 20

H105 29 15 13 8 10 16 7 6 16

H106 101 16 20 15 116 29 16 23 17

H107 52 15 24 10 17 19 12 8 20

H108 33 13 14 10 11 17 30 6 15

H109 57 19 25 27 21 25 41 114 30

H110 50 22 42 21 19 25 10 11 19

H111 32 23 20 15 17 27 98 11 18

H112 69 15 15 16 11 17 6 12 17

H113 91 21 17 21 23 48 40 13 20

H114 94 32 24 27 16 33 10 12 26

H115 138 19 42 33 75 34 13 15 21

9

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FIGURE LEGENDS 1

2

Figure 1 – Cluster analysis of screening data. Heat map overview of log10-3

transformed MFI data from the bead based screening of 134 samples (115 sera) 4

against 61 recombinant M. mycoides SC surface proteins and His6ABP. Colour 5

intensity denotes signal intensity. Proteins to which high amounts of serum antibodies 6

were detected in CBPP-positive sera, thus suggesting a diagnostic importance, formed 7

a cluster of nine proteins (top vertical dendrogram, the cluster is highlighted in red). 8

Horizontally, sera were separated into five groups. Cluster I, II and V contained a 9

majority of the CBPP-positive samples (red). Cluster III contained all but one 10

hyperimmune sera (yellow) which displayed signals of low intensities, while cluster 11

IV contained mostly CBPP-negative bovine sera (blue) as well as the positive control 12

antiserum to M. mycoides SC PG1. All technical replicates clustered together, except 13

for one specimen of a Swedish negative control serum. 14

15

Figure 2 – Statistical selection of immunogenic antigens. In a display of the result 16

from the significance test of the log10-transformed screening data, eight proteins 17

(black) show signal fold changes between the CBPP-positive and the CBPP-negative 18

populations that are greater than 3 and significant with p-values ranging from 10-6

to 19

10-18

. Additionally four proteins (grey) displayed fold changes with significance 20

exceeding the p-value limit of 0.01, but of less discriminative importance than the top 21

eight. Based on this test, the top eight proteins were selected as candidates for an 22

antigen cocktail ELISA. 23

24

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Figure 3 – Cluster analysis based on selected proteins. Screening data from the 1

eight selected proteins were used to categorize all bovine samples, 117 samples from 2

97 sera. Two major clusters were formed, one predominantly CBPP-positive (dark 3

grey) and one CBPP-negative (light grey). 4

5

Figure 4 – Discriminatory power of selected proteins. The range of signal 6

intensities from the screening of all 52 CBPP-positive sera (grey) and 24 CBPP-7

negative sera (white, swedish control sera excluded) against the eight selected 8

antigens show a large discrepancy between the two groups. Potential binding to the 9

fusion tag was monitored by the His6ABP bead. The boxplot was built directly on 10

signal intensities and the box contains 50 % of the data points (inter quartile range), 11

the black line denotes the median signal and the whiskers extend to the furthest data 12

point unless this is situated further away than 1.5 times the inter quartile range. 13

Outliers are presented as circles. 14

15

Figure 5 - Reproduciblity of the antigen cocktail ELISA. Two independent ELISA 16

experiments with 15 sera in triplicates were performed by different persons one month 17

apart to determine the reproducibility of the assay. The two bars for each sample 18

represents the mean value for each experiment and the dots represents the maximum 19

and the minimum value. All signals were normalised to the reference sera B001 20

resulting in low intra and inter assay variation (average CV 3% and 8% respectively). 21

A percent positivity of 50% was suggested as the diagnostic cut-off, shown by a 22

dashed line. 23

24

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Figure 6 – Proof of concept antigen cocktail ELISA. A) Twenty-four CBPP-1

positive (grey) and 23 CBPP-negative (white) bovine sera were analyzed twice by the 2

antigen cocktail ELISA. The optic density values were transformed to percent 3

positivity in reference to sample B001, and presented as averages from the two 4

experiments. A suggested cut-off value of 50% would result in two false negatives 5

and no false positives, assigning 96% of the tested samples correctly. The average 6

percent positivity from CBPP-positive sera was 105% compared to 22% from CBPP-7

negative sera, thus yielding a positive to negative signal ratio of 4.8. B) Receiver 8

operator characteristics of the ELISA results with an area under the curve (AUC) of 9

0.991 indicate a high diagnostic capability. 10

11

12

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B045B003B038B010B010B044B050B002B001B011B011B011B030B017B017B017B052B047B009B009B013B015B016B046B007B018B042B019B054B023B057B027B025B055B026B022B059B031B056B053B058B024B043B014B020H102H107B098B094B097B096B095H106H111H104H101H103H114H112B084B028H115H113H100H108H109H105H110B082B065B089B083B077B078B078B090B029B079B087B069B081B064B070B066B062B071B073B075B091B079B079B072B032H099B063B076B061B068B021B060B067B033B074B085B080B086B088B093B093B093B093B093B093B092B093B093B093B093B049B048B008B008B008B039B037B041B005B004B006B040B034B035B051B036

R.0

321

R.0

906

R.0

910

R.0

187

R.0

847

R.0

298

R.0

364

R.0

177

R.0

924

R.0

104

R.0

521

R.0

285

R.0

711

R.0

922

R.1

001

R.1

033

R.1

050

R.0

668

R.1

052

R.0

292

R.0

061

R.0

923

R.0

574

R.0

198

R.0

755

R.0

392

R.0

990

R.0

540

R.0

754

R.0

320

R.0

992

R.0

390

R.0

346

R.0

693

R.0

173

R.0

344

R.0

844

R.0

707

R.0

809

R.0

422

R.0

552

R.0

203

His

.ABP

R.0

858

R.0

419

R.0

632

R.0

757

R.0

317

R.0

367

R.0

051

R.0

122

R.0

117

R.0

322

R.1

046

R.0

136

R.0

653

R.0

240

R.0

570

R.0

397

R.0

431

R.0

816

R.0

813

10

10

10

10

MFI [A

U]

Density

12

34

CBPP-p

ositiv

e s

era

CBPP-n

egativ

e s

era

Hyperim

mune s

era

III

III

IV

V

Sera

Proteins

0.40

0.2

0.6

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0.6 1.0 1.6 2.5 4.0 6.3 10 16

05

10

15

Fold change (m / m )

P-v

alu

e (-

log)

R-1046R-0136R-0431R-0653R-0813R-0816R-0240R-0397

R-0051R-0711R-0570 R-0322

P = 0.01

Fold change = 3

pos neg

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Sera

B094

B097

B096

B095

B028

B090

B084

B098

B081

B083

B086

B080

B062

B073

B065

B089

B078

B078

B077

B029

B082

B087

B032

B072

B070

B079

B069

B091

B092

B079

B079

B075

B066

B088

B093

B093

B071

B093

B093

B064

B021

B060

B076

B085

B033

B074

B056

B059

B093

B093

B093

B093

B093

B093

B063

B067

B055

B054

B022

B031

B053

B026

B061

B068

B003

B042

B035

B038

B010

B036

B014

B044

B016

B007

B010

B052

B019

B015

B009

B009

B037

B047

B049

B013

B046

B011

B011

B011

B018

B039

B051

B045

B005

B004

B006

B025

B057

B027

B024

B058

B023

B048

B040

B002

B020

B017

B017

B017

B030

B008

B008

B008

B034

B050

B001

B043

B041

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PB

A.si

H

63

10.

R

04

20.

R

79

30.

R

13

40.

R

35

60.

R

31

80.

R

61

80.

R

64

01.

R

5

10

50

100

500

1000

5000

10000

)U

A( IF

M

Antigens

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B001

B002

B009

B011

B019

B020

B027

B050

B058

B063

B064

B067

B069

B078

B087

bla

nk

Perc

ent

Positiv

ity

0

20

40

60

80

100

120

140

Sera

CBPP-positive sera

CBPP-negative sera

Reference serum

Serum free control

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Tru

e P

ositiv

e R

atio (

Sensitiv

ity)

0.0 0.2 0.4 0.6 0.8 1.0

AUC = 0.991

CBPP-pos CBPP-neg

20

40

60

80

100

120

Perc

ent

Positiv

ity

Serum groups

False Positive Ratio (1 - Specificity)

0.0

0.2

0.4

0.6

0.8

1.0

A

B

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