Early antibody response against <it>Mycobacterium avium </it>subspecies <it>paratuberculosis </it>antigens in subclinical cattle

1477-5956-6-5 1477-5956 Research Early antibody response against <it>Mycobacterium avium </it>subspecies <it>paratuberculosis </it>antigens in subclinical cattle Bannantine P John john.bannantine@ars.usda.gov Bayles O Darrell darrell.bayles@ars.usda.gov Waters W Ray ray.waters@ars.usda.gov Palmer V Mitchell mitchell.palmer@ars.usda.gov Stabel R Judith judy.stabel@ars.usda.gov Paustian L Michael michael.paustian@ars.usda.gov

Bacterial Diseases of Livestock Research Unit, USDA/ARS/National Animal Disease Center, Ames, IA 50010, USA

Proteome Science 1477-5956 2008 6 1 5 http://www.proteomesci.com/content/6/1/5 18226229 10.1186/1477-5956-6-5 17 8 2007 28 1 2008 28 1 2008 2008 Bannantine et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Our laboratories have previously reported on the experimental infection of cattle with Mycobacterium avium subsp paratuberculosis (M. paratuberculosis) using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 M. paratuberculosis coding sequences. These combined tools have enabled a unique look at the temporal analysis of M. paratuberculosis antigens within the native host. The primary objective of this study was to identify M. paratuberculosis antigens detected by cattle early during infection. A secondary objective was to evaluate the humoral immune response in cattle during the initial year of infection.

Results

Sera from two experimentally infected cattle, taken pre-inoculation and at day 70, 194 and 321 post infection, identified dynamic antibody reactivity among antigens with some showing an increased response over time and others showing declining levels of reactivity over the same time period. A M. paratuberculosis specific protein, encoded by MAP0862, was strongly detected initially, but the antibody response became weaker with time. The most reactive protein was a putative surface antigen encoded by MAP1087. A second protein, MAP1204, implicated in virulence, was also strongly detected by day 70 in both cattle. Subsequent experiments showed that these two proteins were detected with sera from 5 of 9 naturally infected cattle in the subclinical stage of Johne's disease.

Conclusion

Collectively these results demonstrate that M. paratuberculosis proteins are detected by sera from experimentally infected cattle as early as 70 days after exposure. These data further suggest at least two antigens may be useful in the early diagnosis of M. paratuberculosis infections. Finally, the construction and use of a protein array in this pilot study has led to a novel approach for discovery of M. paratuberculosis antigens.

Background

Johne's disease is an economically significant intestinal disease caused by Mycobacterium avium subsp paratuberculosis (M. paratuberculosis). A recent survey estimated that 20%–40% of dairy herds in the United States are infected with M. paratuberculosis and producers lose $227 USD annually for each infected animal 1. These costs are mostly attributed to the decreased milk production and weight loss resulting from the disease.

After M. paratuberculosis infection by ingestion of contaminated milk or grass containing fecal material from a shedding cow, there is a lengthy subclinical phase that can last for several years. During this stage the cows may appear healthy, but can intermittently shed low numbers of mycobacteria in the feces, enabling transmission to other animals including wildlife species. A major challenge in controlling Johne's disease is the ability to detect infected cattle prior to appearance of disease signs, such as diarrhea and heavy fecal shedding of M. paratuberculosis. An unknown trigger, possibly stress during lactation or parturition, advances the disease from subclinical to clinical where disease signs such as weight loss and diarrhea become evident 23. This trigger appears to coincide with a shift in immune function from a Th1 response to a Th2 response 4. Current detection of subclinical animals depends on the timing and sensitivity of the test. Even the most sensitive culture-based tests will not detect M. paratuberculosis if a subclinically infected animal is not shedding bacilli at the time the fecal or milk sample is collected. M. paratuberculosis antigen induced interferon (IFN)-γ has been shown to be elevated in subclinical animals, but this cytokine declines in the clinical stage concomitant with an increase in M. paratuberculosis specific IL-10 production 56. A comprehensive cytokine profile has been reported for both circulating monocytes and local tissues obtained from M. paratuberculosis-infected cattle 7.

With a few notable exceptions 8910, there is very little data on antibody detection of M. paratuberculosis at early stages of infection in cattle. There are several reasons for this, but one in particular is that cattle that appear healthy are not routinely evaluated using serial test bleeds and analysis. Furthermore, there are numerous studies that show the cell-mediated immune response in cattle predominates during the early stages of infection and is responsible for the initial control of this infection 4611. However, despite the lack of data describing the temporal detection of specific antigens by host antibodies early post infection, these experiments are critical to gain a better understanding of the pathogenesis, diagnostics and vaccine strategies for Johne's disease. For example, the ideal diagnostic antigen would be detected early and remain easily detected throughout the course of the disease. Alternatively, a good vaccine candidate antigen might only be detectable by antibody at a specific stage of the disease. Thus far, no such antigen has been discovered for Johne's disease.

The recent literature has revealed an emphasis on developing consensus animal models for Johne's disease study. One publication 12 is a result of an international meeting, sponsored by the Johne's Disease Integrated Program in the United States, which had the goal of proposing consensus animal models for each of the commodity groups including sheep and cattle as well as farmed deer in New Zealand. Detailed methods including dose, route, length of time for disease signs to appear, etc. were determined 12. A second communication, published independently around the same time, provided similar information 13. Prior to these comprehensive animal model reviews, we published a study describing the intratonsillar route of infection with M. paratuberculosis in neonatal calves 9. Serial test bleeds were collected over the course of the 321-day study and analyzed for T cell and B cell responses using a variety of immunological assays. Selected test bleeds from that study were used on the protein array in the current study. The health status of these calves was further monitored using additional tests, including fecal culture and IS900 PCR. Although M. paratuberculosis was detected in all calves by 271 days post challenge, no clinical signs were observed in any animal throughout the study 9. However, an antibody response was observed by 134 days post infection using a lipoarabinomannan-based ELISA test. A second study that evaluated an experimental infection of goats observed an antibody response as soon as 180 days post infection 14.

The study described herein combines the intratonsillar infection model 9 with newly developed protein array tools to obtain a temporal picture of antigen detection during the initial year of infection in cattle. While the concept of protein arrays has been around nearly as long as the DNA array, there are very few protein arrays available. This is because the process of constructing these arrays involves the production of dozens of specific proteins, a rate limiting and labor-intensive step when compared to oligonucleotide synthesis used in constructing DNA arrays. Nonetheless, studies using bacterial protein arrays are beginning to appear in the literature 151617 due to the power provided by this tool for parallel protein analyses. Our laboratory initially developed a 48 spot protein array consisting of purified recombinant proteins representing selected M. paratuberculosis gene products 18. That array was expanded to a 96-spot array and used in this study.

Results

Construction of a partial M. paratuberculosis protein array

A 96-spot protein array was constructed on nitrocellulose filters using a dot blot apparatus to imprint the array. Represented among these spots are 92 M. paratuberculosis recombinant fusion proteins including hypothetical proteins (20), known antigens (6), membrane proteins (10), and proteins with no similarity in public sequence databases (25). The affinity tag present within these recombinant fusion proteins is the maltose binding protein (MBP). MBP was also expressed as a fusion with the E. coli LacZ alpha peptide and used as a control to determine antibody reactivity to this tag in each experiment. In addition, there are two spots that contain a sonicated whole cell extract of M. paratuberculosis and one spot with only spotting buffer as a negative control. A complete listing of the proteins present on the array along with their sizes, concentration and predicted function are listed in Table 1.

Table 1

Mycobacterium avium subsp paratuberculosis proteins used in this study

Protein name^a

Concentration (mg/ml)^b

Gene size^c

Amino acids^d

Predicted function

MAP sonicate

1.0

NA^e

MAP0075

0.18

423

140

conserved small membrane protein

MAP0087-his

1.0

717

238

probable secreted protein

MAP0105c

1.24

1058

352/889

hypothetical protein

MAP0182c

0.67

411

136

conserved hypothetical protein

MAP0216

1.66

1028

342/347

antigen 85A, mycolyltransferase

MAP0261c

0.49

465

154/161

19 kDa protein

MAP0389

2.04

947

315/337

diarylpropane peroxidase (EC 1.11.1.14) [Nostoc]

MAP0736

5.73

744

247

hypothetical protein

MAP0852

10.4

546

181

no BLAST hits

MAP0853

4.04

660

219

no BLAST hits

MAP0855

0.34

926

308/314

no BLAST hits

MAP0857c

0.70

318

105

hypothetical protein

MAP0858

0.44

537

178/182

no BLAST hits

MAP0859c

0.66

609

202

Mycobacterium phage L5

MAP0860c

0.04

885

294/296

no BLAST hits

MAP0861

0.43

342

113

no BLAST hits

MAP0862

0.48

989

329/360

no BLAST hits

MAP0863

0.66

675

224

no BLAST hits

MAP0864

7.42

426

141

no BLAST hits

MAP0865

0.08

1272

423

Hypothetical 30.9 kDa protein

MAP0866

3.07

804

267

integrase

MAP0904

0.81

371

124/246

conserved hypothetical protein

MAP0961c

0.18

719

239/352

hypothetical protein

MAP1087

0.43

441

146

prob. peptide transp. system permease, N-terminal

MAP1121c

0.32

498

165

putative substrate binding protein

MAP1174c

1.64

756

251

glucose-6-phosphate 1-dehydrogenase

MAP1204

2.61

735

244

putative exported p60 protein homologue

MAP1233

0.04

723

240

conserved hypothetical protein

MAP1345

0.69

600

199

no BLAST hits

MAP1388

0.08

396

131

no BLAST hits

MAP1416c

0.45

666

221

no BLAST hits

MAP1417c

0.54

435

144

no BLAST hits

MAP1609c

0.05

987

328/330

antigen 85B, mycolyltransferase

MAP1636c

1.12

425

141/157

no BLAST hits

MAP1643

0.45

2277

759/762

isocitrate lyase, [beta] module

MAP1655c

0.93

381

126

hypothetical protein

MAP1730c

0.26

1023

340

putative ATP/GTP-binding protein

MAP1931c

0.32

1104

367

anthranilate phosphoribosyltransferase

MAP2077c

1.66

330

109

hypothetical protein

MAP2116c

0.27

1269

422

cell invasion protein

MAP2121c

7.14

887

295/307

major membrane protein I

MAP2121c-his

0.99

924

307

major membrane protein I

MAP2151

2.79

438

145

no BLAST hits

MAP2155

9.57

312

103

similar to IS6110

MAP2156

0.59

963

320

putative transposase GEN: X52471)

MAP2157

0.38

1221

406

IS900

MAP2158

2.40

582

193

no BLAST hits

MAP2182c

0.26

435

144

conserved hypothetical protein

MAP2231

0.31

1422

473

PKS-associated protein, unknown function

MAP2360c

0.13

534

177

hypothetical protein

MAP2380

2.00

600

200/550

acyl-CoA synthase

MAP2657

0.26

651

216

putative oxidoreductase

MAP2663c

0.03

510

169

putative integral membrane protein

MAP2676c

1.30

354

117

hypothetical protein

MAP2734

0.37

1341

446

putative dioxygenasesdiooxygenases

MAP2740

0.24

666

221

putative membrane protein

MAP2751

0.70

582

193

no BLAST hits

MAP2753

0.17

309

103/252

hypothetical protein

MAP2761c

0.03

692

230/238

no BLAST hits

MAP2762c

1.45

425

141/146

no BLAST hits

MAP2764c

0.10

290

96/149

no BLAST hits

MAP2765c

0.06

539

179/396

no BLAST hits

MAP2767c

0.42

473

157/183

no BLAST hits

MAP2963c

0.88

1499

499/874

no BLAST hits

MAP3084c-his

1.0

684

227

probable secreted protein

MAP3121

0.38

855

284

enoyl-CoA hydratase/isomerase superfamily

MAP3129

0.09

404

134/141

hypothetical protein

MAP3131

0.30

1096

365/942

conserved large membrane protein

MAP3155c

1.22

273

hypothetical protein

MAP3434

0.18

690

230/330

possible membrane protein

MAP3437c

0.29

843

279/280

no BLAST hits

MAP3531c

0.28

1034

344/352

antigen 85C, mycolytransferase

MAP3734c

0.19

1559

519/593

putative ABC transporter ATP-binding protein

MAP3735c

0.42

1181

393/429

part of heavy metal tolerance protein

MAP3743

0.85

998

332/348

hypothetical protein

MAP3751

0.11

879

293/979

conserved large membrane protein

MAP3753

0.64

1353

450

hypothetical protein

MAP3761c

0.07

729

242

conserved membrane protein

MAP3771

0.02

294

50S ribosomal protein L31

MAP3817c

0.65

939

312

no BLAST hits

MAP3833c

0.13

626

208/260

hypothetical protein

MAP3840

0.10

1872

623

70 kD heat shock protein, chromosome replication

MAP3902c

0.15

522

173

Serine/threonine kinase (EC 2.7.1.37) [Myc...

MAP3903c

0.42

531

176

Serine/threonine kinase (EC 2.7.1.37) [Myc...

MAP3954

0.05

279

conserved hypothetical protein

MAP4014

0.21

951

316

probable 4-amino butyrate transporter, N-terminal

MAP4025

0.32

854

284/325

cytochrome c-type biogenesis protein

MAP4129

0.26

965

321/336

ABC transporter

MAP4198

0.13

1283

427/441

SecY subunit of preprotein translocase

MAP4207c

5.28

686

228/231

probable ATP-binding transport protein

MAP4199

0.03

540/546

179/181

probable adenylate kinase

MAP4228

0.05

222

initiation factor IF-1

^aProtein name corresponds to the genome project designation (Li et al., 2005). A second designation is included if known.

^bProtein concentration is reported in as measured by Nanodrop spectrometry at 280_nm.

^cGene size is reported in base pairs (bp). In cases where only a portion of the M. avium subsp paratuberculosis gene was cloned, the number of bp present in the clone is reported first followed by the number of bp in the full-length M. avium subsp paratuberculosis gene.

^dNumber of M. avium subsp paratuberculosis amino acids present in the fusion protein. In cases where only a portion of the M. avium subsp paratuberculosis protein is represented, the number of amino acids present is listed first followed by the total number of M. avium subsp paratuberculosis amino acids in the full-length protein.

^eNA is not applicable.

The performance of the array was tested using a monoclonal antibody developed to the MBP tag as shown previously 18. These experiments showed that all proteins containing the tag were readily detectable by immunoblot analysis (data not shown).

Humoral immune response of experimentally infected cattle

An analysis of serial bleeds collected from two experimentally infected calves was performed to temporally examine the humoral immune response to the arrayed M. paratuberculosis proteins. In general, the antibody response against the set of 92 proteins was remarkably similar between the two animals. One notable difference was that fewer proteins were recognized at day 70 in animal 5904 compared to 5902, as only 11 proteins compared to 27 proteins were detected, respectively (Figure 1 and Figure 2). However by day 194, animal 5904 had antibodies against more M. paratuberculosis proteins than did animal 5902. Serum from both animals detected similar numbers of proteins at the end of the study period (Figure 1). The pre-infection sera showed no detection of any of the recombinant proteins (Figure 2) and there was no detection of the MBP-LacZ protein for all time points suggesting that these calves do not have antibodies to the maltose binding protein (MBP) affinity purification tag.

Figure 1

Total number of M. paratuberculosis recombinant proteins detected by sera from experimentally infected cattle

Total number of M. paratuberculosis recombinant proteins detected by sera from experimentally infected cattle. The graph shows the number of proteins on the 96-dot array (y-axis) that were detected at the day post infection (x-axis) for each calf. The total number of proteins detected is indicated on each bar. Note that similar numbers of proteins were detected by each animal at the end of the study period.

Figure 2

Use of the dot blot protein array to obtain antibody reactivity profiles of experimentally infected calves

Use of the dot blot protein array to obtain antibody reactivity profiles of experimentally infected calves. Shown are the spot assignments for the array (A) and dot blot arrays exposed to sera from experimentally infected calves (B). The time point for when each serum sample was collected is indicated in the margin beneath the images. The animal number is listed in the right margin. A whole cell lysate representing a majority of the proteins produced by M. paratuberculosis is spotted in E12 and H12 for all dot blots. Three proteins present on the upper right corner of the array (in column 12) are polyhistidine tagged proteins (MAP0087, MAP2121c and MAP3084c). The remaining 89 spots contain MBP fusion proteins of M. paratuberculosis coding sequences. Note that the MAP2121c coding sequence is represented twice on the array; once as an MBP fusion (spot F4) and also a polyhistidine tagged protein (12B).

Temporal analysis of M. paratuberculosis proteins

The arrays exposed to cattle sera were analyzed by densitometry to obtain a quantitative measure of antibody reactivity (Table 2). When evaluating selected proteins over the course of the study, certain trends in reactivity appear to emerge for some proteins. For example, in both calves, antibody reactivity declined between day 194 and day 321 for a group of 11 proteins. This was especially true for MAP0862 and MAP2657, which showed the most severe decline among these 11 proteins (Table 2). The seven proteins that best fit into strong, moderate and weak response categories are shown in Figure 3. While strong and moderately detected proteins showed a wide range between the calves at days 70 and 194, this range was considerably reduced in the high category proteins at day 321 (Figure 3).

Figure 3

Temporal trends of protein reactivity in experimentally infected cattle

Temporal trends of protein reactivity in experimentally infected cattle. The lines show the average intensity for each protein over the course of the study period. The range observed between the two calves is indicated by statistical bars. Kernel Density Estimation performed on the log-transformed maximum intensity scores indicated there were three distinct normally distributed peaks. These distributions are statistically significant and define the proteins belonging to the weak, moderate, or strong response groups. The graph labeled high contains the seven proteins most strongly detected by the cattle sera and the graph labeled low contains the seven proteins that showed the least reactivity with cattle sera. The seven proteins in the medium category are also representative of reactive antigens that either showed low reactivity initially or declining reactivity with time.

Table 2

Spot intensities of proteins present on the array.^a

Cow 5902

Cow 5904

Spot Loc^b

Protein

Day 0

Day 70

Day 194

Day 321

Day 0

Day 70

Day 194

Day 321

MAP0075

4.472

21.431

21.122

27.612

21.36

A12

MAP0087-his

0.978

1.112

1.882

1.228

MAP0105c

6.929

3.867

5.096

1.186

2.321

10.106

1.813

MAP0182c

5.407

1.924

1.177

2.409

1.617

22.366

MAP0216

0.652

3.779

1.296

1.395

2.175

3.444

MAP0261c

2.395

2.287

3.487

5.668

1.097

3.146

2.397

2.496

MAP0389

7.809

3.163

1.817

1.533

16.751

5.529

MAP0736

0.734

1.446

MAP0852

2.257

3.747

2.908

1.147

2.564

4.661

2.709

1.537

MAP0853

2.144

3.734

5.095

8.497

1.961

4.158

3.239

2.649

MAP0855

2.538

3.387

4.552

7.898

1.716

2.248

2.132

MAP0857c

2.709

6.445

21.867

37.718

1.045

1.602

23.425

15.825

MAP0858

3.005

3.882

1.775

3.999

1.978

MAP0859c

2.821

4.137

2.87

3.745

1.909

2.803

MAP0860c

2.996

5.531

12.743

1.592

7.669

6.676

MAP0861

5.074 ^c

3.407

3.053

1.546

4.05

3.111

3.52

MAP0862

1.795

40.036

16.196

7.997

2.796

11.451

30.918

12.422

MAP0863

1.518

5.524

3.174

4.592

2.057

4.891

3.274

1.473

MAP0864

1.255

3.44

1.781

1.726

2.016

3.56

MAP0865

1.909

18.708

32.138

44.846

1.317

4.386

31.361

35.251

MAP0866

2.346

7.051

2.67

7.116

1.315

3.561

4.052

6.009

MAP0904

1.678

MAP0961c

1.797

14.366

26.5

37.546

0.78

2.103

33.046

42.877

A11

MAP1087

0.753

53.179

51.634

70.968

0.874

33.469

75.392

68.964

B11

MAP1121c

0.758

2.438

2.094

0.699

3.364

1.848

A10

MAP1174c

2.301

3.155

4.411

0.623

1.921

5.81

4.955

C11

MAP1204

0.78

40.8

43.856

55.046

28.367

61.122

58.985

MAP1233

2.568

12.375

22.507

32.493

1.932

3.212

24.157

30.447

MAP1345

3.339

1.861

1.018

1.147

3.413

2.389

MAP1388

2.281

0.429

2.835

MAP1416c

1.647

0.791

3.791

MAP1417c

1.052

14.882

4.095

6.212

0.955

3.979

7.614

3.521

MAP1609c

0.733

3.916

1.003

5.292

MAP1636c

3.286

6.944

13.249

17.272

1.701

17.226

12.06

MAP1643

4.155

1.534

1.827

MAP1655c

2.788

2.049

MAP1730c

1.147

29.831

43.872

52.35

12.272

57.799

57.47

B10

MAP1931c

2.917

2.217

0.511

2.886

2.077

3.339

MAP2077c

3.833

2.784

9.443

1.385

3.63

9.221

D11

MAP2116c

1.418

29.88

33.215

42.633

0.568

11.24

51.336

47.918

MAP2121c

1.76

4.158

1.373

1.513

0.969

3.005

B12

MAP2121c-his

1.247

0.889

2.172

21.226

2.155

MAP2151

4.136

1.919

12.688

21.897

1.571

3.402

24.619

20.889

F12

MAP2155

2.434

1.385

1.251

2.039

2.105

G12

MAP2156

3.731

2.327

2.12

1.357

2.647

5.42

5.113

MAP2157

3.504

0.751

1.532

1.51

2.683

MAP2158

1.403

3.487

4.2

3.396

3.187

1.704

6.682

2.204

MAP2182c

19.101

7.349

8.933

27.935

12.096

E11

MAP2231

1.846

3.732

11.54

15.671

1.166

17.203

18.933

MAP2360c

3.168

2.766