Veterinary Immunology and Immunopathology 157 (2014) 12–19

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Research paper

T lymphocyte responses during early enteric Mycobacterium avium subspecies paratuberculosis infection in cattle Brandon L. Plattner ∗ , Elise Huffman, Douglas E. Jones, Jesse M. Hostetter Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA, USA

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Article history: Received 23 January 2013 Received in revised form 25 October 2013 Accepted 1 November 2013 Keywords: Bovine Lymphocytes Immunology Paratuberculosis Johne’s disease Innate immunity Subclinical

a b s t r a c t Johne’s disease (JD) is a costly intestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (Map), which is transmitted to perinatal calves by the fecal-oral route. Disease control efforts focus on identification and culling of infected cattle from herds; therefore failure to identify animals early is a major obstacle to reducing transmission. Development of host immunity during early JD remain incompletely characterized so detecting subclinical JD using immunologic techniques is a substantial challenge in the field. Development of a test with high sensitivity and specificity is a major research goal with significant implications for the cattle industry. The objectives of this study were to compare early Map-specific T lymphocyte responses in naive, experimentally Map infected and Map vaccinated calves using a subcutaneous matrigel biopolymer-based assay. We examined the phenotype of recruited lymphocytes and local interferon gamma (IFN␥) production within subcutaneously placed matrigel containing Map antigen 30 days after experimentally induced intestinal Map infection or Map vaccination. We show that IFN␥-secreting CD4+ T cells are recruited to matrigel sites in vaccinated but not infected or naïve calves. ␥␦ T cells recruited to matrigel sites of Map-infected calves were mostly WC1-, while ␥␦ T cells recruited to matrigel sites of Map-vaccinated calves were predominantly WC1+. IFN␥ at matrigel sites was a discriminating factor between infected calves, naïve calves and vaccinated calves. These data contribute to our understanding of early anti-Map immunity, and may be useful for detecting early intestinal Map infections in calves or for enhancing our ability to discriminate between Map-infected and Map-vaccinated calves. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Mycobacterium avium subspecies paratuberculosis (Map) is the causative agent of Johne’s disease (JD), a devastating chronic intestinal inflammatory disease affecting ruminants worldwide. Estimates of prevalence in North America vary depending on the choice of diagnostic test; however, recent estimates suggest that Map-infected cattle cost the dairy industry millions of dollars annually due

∗ Corresponding author. Current address: Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1. Tel.: +1 519 824 4120x54766; fax: +1 519 824 5930. E-mail address: [email protected] (B.L. Plattner). 0165-2427/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2013.11.001

to reduced milk yield, premature culling, reduced slaughter value and increased risk of infection for susceptible herdmates (Nielsen and Toft, 2008). Calves are typically infected by the fecal-oral route, and the organism is thought to be endocytosed by epithelial M cells overlying ileal Peyer’s patches and then phagocytized by subepithelial antigen presenting cells (Weiss et al., 2006). An early T-helper 1 (Th1)-like immune response is characterized by production of proinflammatory cytokines and mediators such as interleukin-12 (IL-12), interferon-gamma (IFN␥) and nitric oxide (Coussens et al., 2004; Nielsen and Toft, 2008). In animals that fail to clear the initial infection, a lengthy 2–5 year subclinical phase occurs. Eventual loss of Map-specific IFN␥-producing CD4+ T cells correlates with production of nonprotective Map-specific antibodies and clinical disease

B.L. Plattner et al. / Veterinary Immunology and Immunopathology 157 (2014) 12–19

characterized by multibacillary granulomatous enteritis, progressive fecal bacterial shedding, diarrhea, wasting and death (Sohal et al., 2008). Successful management of JD depends on elimination of Map-infected individuals posing a transmission risk to uninfected herdmates; therefore, identifying animals prior to the onset of fecal shedding is crucial. The most widely used diagnostic tests for this disease, fecal culture and serum or milk antibody enzyme-linked immunosorbent assay (ELISA) both have significant limitations during early Map infection, which substantially limit their field utility (Nielsen and Toft, 2008). Measuring Map specific cell-mediated immunity has been proposed as an attractive alternative for detecting responses to Map earlier, though specificity to Map remains a significant challenge (Mikkelsen et al., 2011). Furthermore, strong persistent cell-mediated immunity is induced in animals vaccinated against Map (Platt et al., 2006; Stabel et al., 2011). Therefore, high specificity is critical in order to distinguish infected from vaccinated animals, and from those exposed to related agents such as nonpathogenic environmental mycobacteria or pathogens such as M bovis. Improved diagnostic testing for early Map infection is a major research objective, and improved understanding of host immunity during early Map infection is expected to benefit this effort. We have previously developed an experimental intestinal Map infection model in calves that mimics many features of early infection (Plattner et al., 2011). We also recently described a subcutaneous matrigel-based approach to simultaneously examine multiple parameters of the host immune response during early Map infection of calves (Plattner et al., 2012). The current study was designed to use the subcutaneous matrigel assay to (1) investigate T lymphocyte responses during early intestinal Map infections in young calves and to (2) explore how such responses may be useful for distinguishing calves with enteric Map infection from naïve or vaccinated calves.

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2.3. Bacterial inoculum Map strain K10 bacteria were harvested from stock cultures maintained in our laboratory. Inocula were prepared as previously described (Plattner et al., 2011). 2.4. Animal inoculation and vaccination Animals were surgically inoculated with Map strain K10 bacteria suspended in 250 ␮L isotonic saline solution directly into the distal ileal Peyer’s patches as previously described; the dose (107 cfu Map) and euthanasia time after infection (30 days) were chosen for this study based on previous work with this model (Plattner et al., 2011). For Map vaccination or control calves, 0.5 mL Mycopar® or 0.9% isotonic saline were administered subcutaneously in the left cervical neck region, also 30 days prior to euthanasia. 2.5. Generation of whole cell sonicate (WCS) Growth phase Map strain K10 bacteria were pooled and sonicated on ice with a probe sonicator. Sonication consisted of three cycles of 10 min bursts (18 W) separated by10 min chilling periods. Debris was removed by centrifugation and protein concentration was determined using the Pierce BCA protein assay (Pierce Biotechnology, Rockford, IL). WCS was streaked onto blood agar to confirm purity from bacterial contamination prior to use. 2.6. Matrigel Becton Dickinson Matrigel Basement Membrane Matrix (MatrigelTM , BD Biosciences, Bedford, MA) is a purified solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma. Matrigel was stored at −20 ◦ C and thawed at 4 ◦ C by placing on ice 24 h prior to use. 2.7. Subcutaneous matrigel assay

2. Materials and methods 2.1. Animals All animal protocols were approved prior to the study by the Committee on Animal Care and Use at Iowa State University. Ten (10) four to six week old castrated male Holstein calves were acquired from the Iowa State University dairy research farm (Ames, IA), a herd certified free of Map infection. Calves were individually housed in biosafety level II animal care facility and were maintained four at a time for logistical purposes. Animals were divided randomly into the following groups: uninfected/naive (n = 2), Map infected (n = 4) and Map vaccinated (n = 4).

2.2. Vaccine Mycopar® , a whole-cell bacterin containing inactivated Mycobacterium paratuberculosis in oil (Boehringer Ingleheim Vetmedica, St Joseph MO), was used in this experiment.

Local immune responses in calves were evaluated using the subcutaneous matrigel assay. At 28 days post infection, Map antigen (live Map, 5 × 105 CFU or Map WCS, 5 ␮g) or an equal volume of sterile saline was added to aliquots of freshly thawed matrigel. The final injection volume was adjusted to 500 ␮L matrigel, and injected subcutaneously in the right cervical neck region using a sterile 1.0 mL tuberculin syringe. Matrigel sites were harvested 48 h after matrigel injection, which corresponded to 30 days after the calves were experimentally infected with Map (PID 30). 2.8. Collection of subcutaneous matrigel assay sites for flow cytometry and culture During necropsy examination, matrigel was visually identified in the subcutaneous space through an 8–10 cm skin incision. Matrigel plugs were removed, placed into 5 mL cold sterile isotonic saline to allow matrigel depolymerization, and transported to the laboratory. The volume of matrigel recovered (with recruited cells) in calves varied between 250 ␮L and 1 mL, and was the largest for

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B.L. Plattner et al. / Veterinary Immunology and Immunopathology 157 (2014) 12–19

Table 1 Antibodies for flow cytometry. Primary

Catalog Number

Isotype

Secondary (zenon system)

Isotype

␥␦ TCRa WC1 (n2)a CD4a CD8␤a

GB21A BAQ4A CACT138A BAT82A

IgG2b IgG1 IgG1 IgG1

AF 488b rPEb rPEb rPE/AF610b

IgG2b IgG1 IgG1 IgG1

a b

VMRD Inc, Pullman, WA, USA. Zenon Labeling technology kits, Invitrogen Molecular Probes Inc, Carlsbad, CA, USA.

vaccinated calves and smallest for naïve calves. Following centrifugation, supernatants were collected and stored at −20 ◦ C; cells were collected and counted using a Beckman Coulter counter (Beckman Coulter Inc, Miami, FL). Cells were resuspended in PBS at a final concentration of 1 × 106 cells per 100 ␮L for antibody staining. Following antibody staining, cells were analyzed by flow cytometry; prior to analysis, non-viable cells were removed from final analysis using electronic gating (data not shown), and data were expressed as a percentage of live cells at each matrigel site.

2.13. Histopathology and acid-fast staining

2.9. Monoclonal antibodies

DTH was assessed immediately prior to euthanasia by measuring the skin pinch thickness following intradermal injection of 0.1 mL of purified protein derivative (Johnin PPD = 4.6 mg/mL, National Veterinary Services Laboratories, Ames, IA) in the caudal fold of the tail 72 ± 6 h prior to euthanasia. IFN␥ ELISA assay was performed on whole blood obtained via jugular venipuncture, according to the manufacturer instructions (M bovis IFN␥ test kit for cattle, Bovigam® , Prionics USA, LaVista, NE) with one modification: in place of the kit-supplied M bovis antigen, Map WCS antigen (strain K10, 10 ␮g/well) was added to appropriate wells for stimulation antigen.

Mouse anti-bovine monoclonal antibodies used to characterize T cell phenotype by multi-color flow cytometry are shown in Table 1. Prior to staining, primary antibodies were individually labeled using Zenon immunolabeling technology (Table 1) according to the manufacturers instructions (Invitrogen/Molecular Probes Inc, Carlsbad CA USA). Stained cells were stored in 2% paraformaldehyde solution prior to flow cytometry. 2.10. Flow cytometric data collection and analysis Data were collected using a FACSCanto flow cytometer (Becton Dickinson Biosciences, San Jose, CA), and FlowJo cell analysis software (Tree Star Inc, San Carlos, CA) was used to analyze data. 2.11. Luminex® Immunoassay Matrigel supernatants were incubated with agitation for 2 h at room temperature, then overnight at 2–8 ◦ C with mouse anti-bovine IFN␥-coupled Luminex® beads. After addition of detection antibodies (biotin-labeled mouse anti-bovine IFN␥; streptavidin-PE, Serotec Inc), mean fluorescence intensities of individual samples (in duplicate) were compared to standard curve to determine cytokine concentrations.

Formalin-fixed tissues were paraffin embedded, sectioned at 4 ␮m and stained with hematoxylin/eosin (HE) and ZN acid-fast technique for routine histopathologic evaluation and identification of acid fast organisms, respectively. 2.14. Delayed-type hypersensitivity (DTH) reaction; IFN assay

2.15. Statistical analysis Statistical analysis was performed using JMP 10.0.0 (SAS Institute, Cary, NC). Data are presented as the mean values ± standard error of the mean (SEM). Student’s t-test and one-way analysis of variance (ANOVA) were used for the statistical analysis, unless otherwise specifically stated. Group mean differences were considered significant if the p value was