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Host Responses to Persistent Mycobacterium avium Subspeciesparatuberculosis Infection in Surgically Isolated Bovine Ileal Segments

Chandrashekhar Charavaryamath,a* Patricia Gonzalez-Cano,a Patrick Fries,a* Susantha Gomis,b Kimberley Doig,a Erin Scruten,a

Andrew Potter,a Scott Napper,a,c Philip J. Griebela,d

VIDO/Intervac, University of Saskatchewan, Saskatoon, SK, Canadaa; Department of Veterinary Pathology, WCVM, University of Saskatchewan, Saskatoon, SK, Canadab;Department of Biochemistry, University of Saskatchewan, Saskatoon, SK, Canadac; School of Public Health, University of Saskatchewan, Saskatoon, SK, Canadad

A lack of appropriate disease models has limited our understanding of the pathogenesis of persistent enteric infections with My-cobacterium avium subsp. paratuberculosis. A model was developed for the controlled delivery of a defined dose of M. aviumsubsp. paratuberculosis to surgically isolated ileal segments in newborn calves. The stable intestinal segments enabled the char-acterization of host responses to persistent M. avium subsp. paratuberculosis infections after a 9-month period, including ananalysis of local mucosal immune responses relative to an adjacent uninfected intestinal compartment. M. avium subsp. paratu-berculosis remained localized at the initial site of intestinal infection and was not detected by PCR in the mesenteric lymph node.M. avium subsp. paratuberculosis-specific T cell proliferative responses included both CD4 and �� T cell receptor (��TcR) T cellresponses in the draining mesenteric lymph node. The levels of CD8� and ��TcR� T cells increased significantly (P < 0.05) inthe lamina propria, and M. avium subsp. paratuberculosis-specific tumor necrosis factor alpha (TNF-�) and gamma interferonsecretion by lamina propria leukocytes was also significantly (P < 0.05) increased. There was a significant (P < 0.05) accumula-tion of macrophages and dendritic cells (DCs) in the lamina propria, but the expression of mucosal toll-like receptors 1 through10 was not significantly changed by M. avium subsp. paratuberculosis infection. In conclusion, surgically isolated ileal segmentsprovided a model system for the establishment of a persistent and localized enteric M. avium subsp. paratuberculosis infectionin cattle and facilitated the analysis of M. avium subsp. paratuberculosis-specific changes in mucosal leukocyte phenotype andfunction. The accumulation of DC subpopulations in the lamina propria suggests that further investigation of mucosal DCs mayprovide insight into host responses to M. avium subsp. paratuberculosis infection and improve vaccine strategies to prevent M.avium subsp. paratuberculosis infection.

Johne’s disease (JD) is a chronic enteric infection of ruminantscaused by Mycobacterium avium subspecies paratuberculosis.

Following a prolonged asymptomatic incubation period, clinicaldisease may develop and is characterized by granulomatous in-flammation of the intestine, severe diarrhea, wasting, loss of pro-duction, and death (reviewed in references 1 and 2). M. aviumsubsp. paratuberculosis infection is one of the most costly diseasesaffecting dairy cattle, with economic losses in the United Statesestimated at US$200 million to US$250 million per year (3). Joh-ne’s disease occurs worldwide. Infections are difficult to diagnoseduring subclinical stages, and infected animals shed the bacteriumintermittently in feces and milk. M. avium subsp. paratuberculosisis a robust organism that can survive under a variety of conditions,and its persistence in the environment and in dairy productsmight be a source of infection for humans (reviewed in reference4). A possible link to Crohn’s disease has been suggested (reviewedin reference 5). There is, however, no definitive evidence implicat-ing M. avium subsp. paratuberculosis as the causative agent ofCrohn’s disease (6).

Newborn calves acquire M. avium subsp. paratuberculosis pri-marily by the fecal-oral route and rarely in utero (7). The primarysite of M. avium subsp. paratuberculosis invasion is thought to bethe terminal small intestine or ileum, with bacterial uptake by Mcells in the Peyer’s patches (PP) (8) or direct infection of absorp-tive epithelium (9, 10). Once M. avium subsp. paratuberculosispasses through the mucosal barrier, it establishes a persistent in-fection in macrophages (reviewed in references 11 and 12). Nu-merous studies have been performed to analyze immune re-sponses following infection with M. avium subsp. paratuberculosis

and to identify potential mechanisms by which this organismevades innate (reviewed in reference 13) and acquired (reviewedin reference 14) immune responses. In vitro analysis of the inter-actions of M. avium subsp. paratuberculosis with myeloid cells andanalysis of M. avium subsp. paratuberculosis-specific immune re-sponses in blood (15, 16) have provided insight into the interac-tion between this organism and immune cells. Differential cyto-kine gene expression in blood leukocytes, mesenteric lymph nodes(mLNs), and PP of cows infected with M. avium subsp. paratuber-culosis revealed significant differences between systemic and mu-cosal immune responses (reviewed in reference 17). Since M.avium subsp. paratuberculosis infection persists in the small intes-tine, understanding of the induction and recruitment of mucosaleffector cells to this region is important for developing vaccinesthat prevent this infection. Current vaccines, which are injected

Received 9 August 2012 Returned for modification 29 August 2012Accepted 27 November 2012

Published ahead of print 5 December 2012

Address correspondence to Philip J. Griebel, [email protected].

* Present address: Chandrashekhar Charavaryamath, College of VeterinaryMedicine, Western University of Health Sciences, Pomona, California, USA; PatrickFries, Centre for Food-Borne and Animal Parasitology, Saskatoon, SK, Canada.

C.C. and P.G.-C. contributed equally to this article.

This article is journal series 647 from VIDO/Intervac, University of Saskatchewan.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/CVI.00496-12

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parenterally, reduce the shedding of M. avium subsp. paratuber-culosis and the onset of clinical disease but do not prevent infec-tion (reviewed in reference 18).

Mice are the predominant animal model used to screen vaccinecandidates for M. avium subsp. paratuberculosis (19–22), but therelevance of mouse models is limited by the absence of M. aviumsubsp. paratuberculosis infection in the small intestine. Further-more, newborn calves are the primary target for M. avium subsp.paratuberculosis vaccination (18), and due to the developmentalimmaturity of their immune systems, mice cannot be used forneonatal vaccination studies. Therefore, an effective model of M.avium subsp. paratuberculosis infection is required to evaluate po-tential vaccine antigens, formulations, and delivery systems fornewborn calves. Temporary ligation of the terminal small intes-tine, or ileum, in 3- to 4-week-old calves has been used to analyzemucosal events during the first 24 h after infection with M. aviumsubsp. paratuberculosis (8, 23). The delivery of a defined dose of M.avium subsp. paratuberculosis to the terminal small intestine wasalso achieved by using intestinal clamps to temporarily occlude asection of ileum (24) in 14- to 21-day-old calves. M. avium subsp.paratuberculosis infection and specific immune responses werethen monitored for as long as 9 months postinfection. More re-cently, an ileal cannulation model that facilitated the repeateddelivery of a defined dose of M. avium subsp. paratuberculosis tothe terminal small intestine was developed for 8- to 9-week-oldcalves (25). These models were limited, however, by variable up-take of M. avium subsp. paratuberculosis from the intestine andshedding of inoculated M. avium subsp. paratuberculosis in feces.

Our objective was to determine whether a model of persistentM. avium subsp. paratuberculosis infection could be developed innewborn calves which would facilitate a comparative analysis ofmucosal immune responses at an infected and a noninfected sitewithin the same animal. We demonstrated previously that it waspossible to maintain a surgically isolated segment of terminalsmall intestine in a relatively stable state for 9 to 11 months (26).This model system was used to deliver a defined dose of M. aviumsubsp. paratuberculosis to the terminal small intestine, and by sub-dividing the intestinal segment into multiple compartments, itwas possible to analyze M. avium subsp. paratuberculosis-specificmucosal immune responses.

MATERIALS AND METHODSAnimals, surgery, and infection with M. avium subsp. paratuberculosis.All experimental protocols were performed according to the guidelinesapproved by the Canadian Council on Animal Care. Protocols for animalhousing, anesthesia, surgery, and postsurgical care have been describedpreviously (26). Briefly, newborn male Holstein calves (10 to 14 days old;n � 6) that had received colostrum were fasted for 12 h prior to surgery. Aparalumbar abdominal incision was made and the terminal small intes-tine exteriorized. A 30-cm intestinal segment, containing continuous PP,was identified 20 cm proximal to the ileocecal fold and was demarcated byplacing two consecutive intestinal clamps at each end of the isolated seg-ment. The intestine was transected between each pair of clamps; ingestawere removed by flushing the segment twice with warm calcium- andmagnesium-free phosphate-buffered saline (PBSA); and then an antibi-otic solution (250 mg metronidazole [Hospira Healthcare Corp., Mon-treal, QC, Canada] and 200 mg enrofloxacin [Baytril; Bayer Inc., Toronto,ON, Canada]) was infused into the lumen of the segment and was left for25 min. The continuity of the intestinal tract was reestablished by per-forming end-to-end anastomoses following alignment of the mesentericand antimesentric borders of the intestine proximal and distal to the iso-lated segment. The antibiotic solution in the segment was removed by

flushing with phosphate-buffered saline (PBS; pH 7.4), and both endswere oversewn. The intestinal segment was subdivided into three equalcompartments with silk ligatures, and each compartment was injectedwith 1 g neomycin (Neomix; Pharmacia & Upjohn Animal Health, Oran-geville, ON, Canada), 1.2 g florfenicol (Nuflor; Merck Canada Inc., Kirk-land, QC, Canada), and 250 mg metronidazole in 5 ml PBSA. The distalcompartment was injected with 1 � 108 to 3 � 108 CFU of M. aviumsubsp. paratuberculosis K10 inoculum in 5 ml of PBS, while the proximalcompartment was injected with 5 ml PBS. The middle compartment pro-vided a space to physically separate the infected and uninfected compart-ments and to minimize the probability of contamination of the uninfectedcompartment with M. avium subsp. paratuberculosis. Intestinal perfusionwas not disrupted throughout the surgery, and there was no edema ornecrosis where the intestine was transected or anastomosed. The intestinalsegment was returned to the abdomen, and the abdominal incision wasclosed with a simple continuous suture pattern (no. 1 Maxon; Covidienplc). The subcutaneous layer was closed with a simple continuous suturepattern using a 2-0 absorbable suture (Monocryl; Ethicon), and the skinwas closed with a simple continuous suture pattern (no. 2 Supramid;Serag-Wiessner KG, Germany). Calves were fed whole milk for 16 weeks,after which they were transitioned over a 2-month period to a diet of grainand hay.

M. avium subsp. paratuberculosis K10 was a generous gift from MarcelBehr (McGill University Health Centre, Montreal, QC, Canada), and K10stock was grown in Difco Middlebrook 7H9 broth (BD Bioscience, Can-ada) before aliquots were frozen for infection experiments. A thawed ali-quot of M. avium subsp. paratuberculosis K10 was first grown on Middle-brook 7H10 agar (BD Bioscience, Canada). One loopful of bacteria wasthen inoculated into Difco Middlebrook 7H9 broth, and liquid cultureswere incubated on a rotary shaker for 5 days at 37°C. All infections wereperformed with M. avium subsp. paratuberculosis passaged less than 4times from the original K10 stock. Cultures were centrifuged at 3,000 � gand 4°C for 15 min in a sterile preweighed 50-ml centrifuge tube. Thesupernatant was discarded, and the bacterial pellet was allowed to dry for30 min in the inverted tube before a final weight was obtained. The weightof the dry bacterial pellet was calculated (by subtracting the tube weightfrom the weight of the tube with the pellet), and the total CFU count wascalculated on the basis of previous CFU titration experiments. Serial di-lutions of the dry pellet were made in Ca2�- and Mg2�-free PBS, platedonto Middlebrook 7H10 agar (BD Bioscience, Canada), and incubated at37°C for as long as 6 weeks to determine viable bacterial colony counts.This titration established that 1 mg dry bacterial pellet was equivalent to1 �107 CFU, and this value was used in preparing M. avium subsp. para-tuberculosis inocula to infect intestinal segments.

Cell isolation procedures. Blood was collected from the jugular veinsof calves 1 day prior to infection with M. avium subsp. paratuberculosisand at monthly intervals postinfection. Peripheral blood mononuclearcells (PBMCs) were isolated by following a previously described protocol(7). Briefly, blood was centrifuged at 1,400 � g for 20 min at room tem-perature before the buffy coat was collected and the cells were resus-pended in 35 ml Ca2�- and Mg2�-free PBS containing 0.1% EDTA. Cellswere layered onto isotonic 54% Percoll (GE Healthcare Bio-Sciences AB,Uppsala, Sweden) and were centrifuged at 2,000 � g for 20 min at roomtemperature. PBMCs at the Percoll-PBS interphase were collected,washed three times with PBS, and resuspended at a final concentration of2 � 106 viable cells/ml in RPMI medium (Invitrogen, Burlington, ON,Canada) containing 10% fetal bovine serum (FBS; Invitrogen) plus anti-biotics and antimycotics (Sigma-Aldrich Canada, Oakville, ON, Canada).

The mLNs draining each intestinal segment were collected based ontheir locations in the mesentery adjacent to the segment. There was usu-ally a single mLN proximal to each segment, and it was therefore assumedthat lymphatic drainage included both the control and M. avium subsp.paratuberculosis-infected compartments. Pericapsular fat was removedbefore the mLN was cut in half and immersed in Ca2�- and Mg2�-freePBS. The tissue was minced finely with a scalpel blade to release single

M. avium subsp. paratuberculosis in Ileal Segments

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cells. The cell suspension was passed through a 40-�m nylon cell strainer(Becton Dickinson Labware, Franklin Lakes, NJ), and cells were washedthree times with PBS containing 0.1% EDTA before being resuspended ata final concentration of 2 � 106/ml in Dulbecco’s modified Eagle medium(DMEM) supplemented with 10% FBS plus antibiotics and antimycotics.

Samples (10 cm) were collected from the control and M. avium subsp.paratuberculosis-infected compartments from each intestinal segment im-mediately after calves were euthanized, and tissues were placed in ice-coldDMEM (Sigma-Aldrich, Canada). Lamina propria leukocytes (LPLs)were isolated from each intestinal sample by using the tissue digestionprotocol previously described by Fries et al. (27), and the resulting cellsuspensions were adjusted to a final concentration of 2 � 106 cells/ml inDMEM supplemented with 10% FBS plus antibiotics and antimycotics.

Assays for detection of M. avium subsp. paratuberculosis-specificresponses. A lysate was prepared with M. avium subsp. paratuberculosisgrown in Middlebrook 7H9 broth. The bacterium was pelleted, and thepellet was resuspended at a final concentration of 1.34 � 108 CFU/ml inPBS. The bacterial suspension was sonicated with a high-intensity ultra-sonic processor (Vibra-Cell, Danbury, CT) at 40% amplitude with a cycleof 8 s on and 8 s off for 10 min. Sonicated M. avium subsp. paratuberculosiswas centrifuged at 12,000 � g for 5 min; the pellet was discarded; and theprotein concentration of the supernatant (M. avium subsp. paratubercu-losis lysate) was measured using a Quant-iT protein assay kit (Invitrogen,Canada). Phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich, Canada)was added to the lysate at a final concentration of 1 mM before aliquotswere stored at �20°C.

PBMCs, mLN cells, and ileal LPLs were cultured in 96-well tissueculture plates (Thermo Fisher Scientific, Rochester, NY) in a final volumeof 200 �l DMEM supplemented with 5% FBS plus antibiotics and anti-mycotics. For each cell type, three replicate cultures were stimulated witheither the medium alone, 1 �g/ml M. avium subsp. paratuberculosis lysate,or 1 �g/ml concanavalin A (Sigma-Aldrich Canada). For the lymphocyteproliferation response (LPR) assay, 2 � 105 cells/well were incubated at37°C under 5% CO2 for 5 days, and during the last 6 h of culture, 20 �l[3H]thymidine (0.4 �Ci per well; GE Healthcare Biosciences, Pittsburgh,PA) was added to each well. Plates were freeze-thawed to lyse cells, whichwere harvested with a microplate cell harvester (model C961961;PerkinElmer, Waltham, MA). Radioactivity was detected using a Top-Count NXT beta scintillation counter (model C9912V1; PerkinElmer,Waltham, MA). Average counts per minute were calculated for each set oftriplicate cultures, and stimulation indices (SIs) were calculated by divid-ing the average cpm for cultures stimulated with the M. avium subsp.paratuberculosis lysate by the average cpm for cells cultured in the mediumalone. For the cytokine secretion assays, 5 � 105 cells/well were incubatedin duplicate cultures and were stimulated either with the medium alone orwith 1 �g/ml M. avium subsp. paratuberculosis lysate. After a 48-h incu-bation period, the culture supernatants were harvested and stored at�20°C.

The concentrations of gamma interferon (IFN-�) and tumor necrosisfactor alpha (TNF-�) in culture supernatants were determined by captureenzyme-linked immunosorbent assays (ELISAs) as described previously(28, 29). Net cytokine secretion was calculated by subtracting the level ofspontaneous cytokine release from cells cultured in medium alone fromthe level of cytokine release induced by stimulation with the M. aviumsubsp. paratuberculosis lysate.

The protocol for PKH cell staining was described previously by Bu-chanan et al. (30). Briefly, mLN cells were washed once in serum freeAIM-V medium (Life Technologies, Grand Island, NY) at a final concen-tration of 2.0 �106 cells/ml, and the cell pellet was resuspended in DiluentC. PKH-26 dye (Sigma-Aldrich, Canada) was added to the cell suspen-sion, and after 5 min, the reaction was stopped by the addition of 1%bovine serum albumin (BSA) for 1 min. Cells were washed three timeswith AIM-V medium supplemented with 10% FBS and 50 �mol 2-mer-captoethanol before 2.0 � 106 cells/well were cultured in 6-well plates(Corning, Corning, NY) either with or without the M. avium subsp. para-

tuberculosis lysate or medium. Cells were cultured for 72 h at 37°C under5% CO2 before labeling with monoclonal antibodies (MAbs) specific forCD4, CD8, and �� T cell receptor (��TcR) (VMRD, Inc., Pullman, WA).Labeling with MAbs was detected using fluorescein isothiocyanate(FITC)-conjugated goat-anti-mouse IgG (Southern Biotechnology, Bir-mingham, AL), and cell labeling was analyzed using a FACSCalibur (Bec-ton Dickinson, Franklin Lakes, NJ) flow cytometer. Cell proliferation wasanalyzed using ModFit LT (Verity Software House, Inc. Topsham, ME)flow cytometry modeling software.

The flow cytometry protocol and reagents for labeling LPLs have beendescribed previously in detail by Fries and Griebel (31). Briefly, LPLs werestained indirectly using fluorochrome-conjugated, isotype-specific goatanti-mouse Ig antisera to detect monoclonal antibodies specific for T cells(for CD3, clone MM1A [32]; for CD4, clone CACT183A [33]; for CD8,clone CACT80C [34]; for ��TcR, clone GB21A [35]). LPLs were distin-guished from epithelial cells by costaining for the leukocyte commonantigen, CD45 (clone CACTB51A [36]) and restricting flow cytometricanalysis to CD45� cells. The total number of CD45� LPLs isolated per 10cm of ileum was calculated by first subtracting the number of B cells(CD21� [clone CC21]) (IgM� [clone PIG45A]), which are derived pri-marily from lymphoid follicles present in the submucosa of the ileum(27). Based on the total number of CD45� LPLs, it was then possible tocalculate absolute numbers for individual T cell subpopulations (CD3,CD4, CD8, and ��TcR T cells) and CD335 (clone MCA23650 NK cells)and CD11c� (clone BAQ153A) myeloid cell subpopulations (CD11b[clone MM10A], CD13 [clone CC81], CD14 [clone MM61A], CD26[clone CC69], CD172a [clone CC149], and CD205 [clone CCD98]). Im-mediately after calves were euthanized, intestinal tissue was collected fromboth the control and M. avium subsp. paratuberculosis-infected compart-ments. The tissue sampled included the entire wall of the intestine and wascut into 4- to 5-mm2 fragments before being immersed in RNAlater (Ap-plied Biosystems) and stored at �80°C. Toll-like receptor (TLR) expres-sion was analyzed by quantitative reverse transcription-PCR (qRT-PCR)using specific primers and protocols described previously by Charavary-amath et al. (26). Briefly, tissue was homogenized in TRIzol reagent (In-vitrogen Canada, Inc., Burlington, ON, Canada); RNA was purified usingRNeasy minicolumns (Qiagen); and the RNA concentration was deter-mined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scien-tific, Wilmington, DE). cDNA was synthesized by reverse transcription of500 ng total RNA using the SuperScript III Platinum system included in atwo-step qRT-PCR kit with SYBR green (Invitrogen Canada Inc.) accord-ing to the manufacturer’s protocol. Each PCR was performed in duplicateby using 25 ng of cDNA amplified with each primer set on the Bio-RadiCycler (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada) with thefollowing parameters: 55°C for 2 min to eliminate carryover dUTP; 95°Cfor 8.5 min; 45 cycles of 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s.qRT-PCR data are expressed as the change in the threshold cycle (CT),which was calculated by subtracting the CT for an individual TLR from theCT for -actin. The CT for -actin differed less than 1 cycle among all thesamples analyzed.

Pathology, histology, and detection of M. avium subsp. paratuber-culosis. Calves were euthanized with an intravenous injection ofEuthanyl (20 ml/45 kg body weight; Bimeda-MTC, Canada) immedi-ately prior to the collection of tissue samples. Surgically created intes-tinal segments, attached mLNs, and adjacent intestine were examinedfor grossly visible lesions and were photographed, and tissue sampleswere fixed in 10% neutral buffered formalin for histopathological ex-amination. The intestinal segment and adjacent ileum were openedalong the mesenteric border, the intestinal contents removed, and themucosa examined. The contents of the intestinal segments were placedin sterile tubes and were processed for culture of M. avium subsp.paratuberculosis. All histology samples were processed, and hematox-ylin and eosin (H&E) staining and acid-fast staining were performed atPrairie Diagnostic Services (Saskatoon, SK, Canada).

Intestinal contents were collected, and a 2-g aliquot was diluted in 35

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ml sterile distilled water, vortexed to disperse particulate material, andthen placed on a mechanical rocker for 30 min. Each sample was allowedto settle for 30 min at room temperature before a 5-ml aliquot was re-moved from the top of the tube and added to 35 ml 0.9% hexadecylpyri-dinium chloride solution. Each sample was incubated overnight at 37°Cbefore centrifugation at 900 � g for 30 min. The supernatant was dis-carded and the pellet resuspended in 1 ml sterile water containing 100�g/ml of amphotericin B before 500 �l was spread onto 7H10 agar plates.Culture plates were sealed in a plastic bag and were incubated at 37°C for4 to 6 weeks.

PCR amplification was used to detect M. avium subsp. paratuberculosisin the paraffin-embedded tissue sections collected from M. avium subsp.paratuberculosis-infected and noninfected ileal compartments (n � 6)and adjacent mLNs (n � 6). PCR analysis of tissue sections was performedby Prairie Diagnostic Services (Saskatoon, SK, Canada). DNAs from par-affin-embedded tissue samples were extracted by using the DNeasy bloodand tissue kit (Qiagen, Toronto, ON, Canada) according to the manufac-turer’s instructions. PCR was then performed using the VetAlert Johne’sreal-time PCR kit specific for M. avium subsp. paratuberculosis (Tetra-core, Inc. Rockville, MD).

Statistical analysis. Absolute numbers of each lymphocyte and my-eloid cell subpopulation in M. avium subsp. paratuberculosis-infected ver-sus noninfected compartments were compared using a t test (GraphPad

Prism, version 5.4; GraphPad Software, San Diego, CA). A t test was alsoused to compare the percentages of CD4, CD8, and ��TcR T cell sub-populations proliferating (by ModFit analysis) in the presence or absenceof the M. avium subsp. paratuberculosis lysate for the four animals display-ing increased LPRs (see Fig. 6). Differences in cytokine secretion wereanalyzed using one-way analysis of variance (ANOVA) and Tukey’s mul-tiple-comparison test. Differences were considered significant when P was�0.05.

RESULTSPostsurgical clinical observation of experimental calves. Fol-lowing the preparation of intestinal segments and M. aviumsubsp. paratuberculosis infection, there were no significantchanges in body temperature for the first 5 days postsurgery.Throughout the remainder of the trial, an animal health techni-cian visually inspected animals daily, and no abnormalities in feedintake or the consistency of feces were observed.

Gross examination and histology of intestinal compart-ments. Physical examination of the serosal and mucosal surfacesof isolated ileal segments (both infected and noninfected com-partments) and the adjacent ileum revealed no gross abnormali-ties (Fig. 1A). Furthermore, at 9 months after surgery, the three

FIG 1 Gross anatomy and histopathology of noninfected and M. avium subsp. paratuberculosis-infected ileal compartments. (A) Gross appearance of anintestinal segment in situ 9 months after infection with M. avium subsp. paratuberculosis. The surgically isolated intestinal segment was subdivided into threecompartments with silk ligatures (red arrows): compartment 1, control (proximal noninfected compartment); compartment 2, interspace (intervening com-partment); compartment 3, M. avium subsp. paratuberculosis (distal M. avium subsp. paratuberculosis-infected compartment). The site where intestine proximaland distal to the isolated segment was anastomosed is visible (blue arrow) in the adjacent ileum. The major demarcations on the ruler are 1 cm apart. (B)Hematoxylin-and-eosin-stained tissue section from a noninfected compartment. (C) Hematoxylin-and-eosin-stained tissue section from an M. avium subsp.paratuberculosis-infected compartment with cellular aggregates in the LP (boxed). (D) Cellular aggregate in the LP of an M. avium subsp. paratuberculosis-infected compartment (boxed in panel C), with eosinophils (arrows) surrounding and infiltrating the cellular aggregate.




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compartments prepared in the ileal segment remained distinct,with the distal end of each compartment demarcated by an in-creased accumulation of material within the lumen of that com-partment (Fig. 1A, arrows). This distal accumulation of materialsuggested that peristalsis persisted in the isolated segment. Exam-ination of three H&E-stained sections from each noninfectedcompartment revealed normal microscopic anatomy of intestinalvilli and no organized cellular infiltration in the lamina propria(LP) (Fig. 1B). In contrast, examination of three tissue sectionsfrom each M. avium subsp. paratuberculosis-infected compart-ment (Fig. 1C) revealed numerous goblet cells, a widening andshortening of intestinal villi, and increased cellularity in the LP.Cellular aggregates were observed in the LP (Fig. 1C inset) of atleast one tissue section from each M. avium subsp. paratuberculo-sis-infected compartment. At a higher magnification, the cellularaggregates were frequently seen to be characterized by a peripheralaccumulation of eosinophils (Fig. 1D). Eosinophils appeared to bemore abundant in the LP of the infected compartment (Fig. 1D)than in the control compartment, but neutrophils were rarely ob-served in either the M. avium subsp. paratuberculosis-infected orthe control compartment. Visible acid-fast bacilli were observedin the LP of an M. avium subsp. paratuberculosis-infected com-partment from only one of six animals (data not shown). No acid-fast bacteria were observed in any of the noninfected compart-ments or in the mLNs associated with isolated intestinal segments(data not shown).

M. avium subsp. paratuberculosis culture from intestinalcontents and PCR detection in tissue. Intestinal contents werecollected from both M. avium subsp. paratuberculosis-infectedand noninfected compartments. Culture of M. avium subsp. para-tuberculosis from intestinal contents was attempted using a sedi-mentation and decontamination protocol, but no visible M.avium subsp. paratuberculosis colonies were detected following a9-week incubation at 37°C. DNA was then extracted from tissuesections cut from the paraffin-embedded tissues collected from M.

avium subsp. paratuberculosis-infected and noninfected ileal com-partments as well as from the associated mLNs. PCR analysis re-vealed that M. avium subsp. paratuberculosis-specific DNA wasdetectable in tissues collected at 9 to 11 months postinfection fromM. avium subsp. paratuberculosis-infected compartments but notfrom adjacent noninfected ileal compartments or from the drain-ing mLNs (Fig. 2).

LPL population changes following M. avium subsp. paratu-berculosis infection. Persistent M. avium subsp. paratuberculosisinfection resulted in significant (P � 0.05) infiltration of leuko-cytes into the LP (Fig. 3). Lamina propria leukocytes (LPLs) werespecifically identified by the expression of CD45, which excludedcontaminating mucosal epithelial cells from flow cytometric anal-ysis of the cell phenotype and enabled the calculation of absoluteleukocyte numbers isolated from each 10-cm piece of intestine.Infection of macrophages in the LP of the ileum was reported earlyduring M. avium subsp. paratuberculosis infection (8), but recentinvestigations have identified diverse myeloid subpopulationswithin the LP of newborn calves (27). Therefore, we investigatedwhether M. avium subsp. paratuberculosis infection altered the

FIG 2 PCR detection of M. avium subsp. paratuberculosis infection in surgi-cally isolated ileal segments and associated lymph nodes. PCR analysis wasperformed for all animals (n � 6) by using DNA isolated from formalin-fixedtissue collected from the M. avium subsp. paratuberculosis-infected and non-infected (control) compartments within each surgically isolated ileal segment.Mesenteric lymph nodes located in the mesentery adjacent to each isolatedintestinal segment were also sampled.

FIG 3 Comparison of LPL population changes in M. avium subsp. paratuber-culosis-infected and noninfected ileal compartments. Enzymatic digestion of10-cm segments of ileum was performed to isolate LPLs, and the total numberof cells isolated was recorded for both M. avium subsp. paratuberculosis-in-fected and noninfected ileal compartments. The total number of LPLs isolat-ed/10 cm of tissue was calculated by multiplying the total number of cellsrecovered by the percentage of CD45� cells. (A) Myeloid cells in the LPLpopulations were identified as CD45� CD11c�, and the frequency of individ-ual myeloid cell subpopulations was analyzed for cells coexpressing majorhistocompatibility complex class II (MHC II), CD11b, CD13, CD14, CD26,CD172a, and CD205. (B) CD45� leukocytes were analyzed for coexpression ofCD3, CD4, CD8, ��TcR, and CD335. The data presented are means and 1 SDfor values for the M. avium subsp. paratuberculosis-infected (filled bars) andnoninfected (open bars) compartments within the same animals (n � 5). *,P � 0.05.




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frequency of specific myeloid subpopulations in the LP. At 9 to 11months postinfection, the number of myeloid cells isolated fromthe LP of M. avium subsp. paratuberculosis-infected compart-ments was significantly (P � 0.05) higher than that from nonin-fected compartments (Fig. 3A). Furthermore, the increase inCD11c� myeloid cells involved both macrophage (CD11b�,CD14�, and CD172�) and dendritic cell (DC) (CD13�, CD26�,and CD205) subpopulations (Fig. 3A). Analysis of total T cell(CD45� CD3�) numbers also revealed a significant (P � 0.05)accumulation of T cells in the LP of M. avium subsp. paratubercu-losis-infected compartments compared to that in adjacent nonin-fected compartments (Fig. 3B). This analysis also revealed thatboth CD8� and ��TcR� cells contributed significantly (P � 0.05)to the increased T cell numbers in the LP of the M. avium subsp.paratuberculosis-infected compartments (Fig. 3B). The numbersof CD4� T cells and CD335� NK cells were not significantlychanged at 9 to 11 months postinfection.

Effect of M. avium subsp. paratuberculosis infection on TLRexpression. Evidence that M. avium subsp. paratuberculosis infec-tion significantly alters LPL myeloid cell populations suggestedthat infection with this bacterium may also alter other aspects ofthe innate mucosal immune system. Therefore, we compared theexpression of tlr-1 through tlr-10 in M. avium subsp. paratubercu-losis-infected versus noninfected ileal compartments. No signifi-cant differences in tlr expression were detected 9 to 11 months

following M. avium subsp. paratuberculosis infection by usingRNA extracted from ileal tissue, which included the mucosa, LP,submucosa, and serosa (Fig. 4).

M. avium subsp. paratuberculosis-specific LPRs and cyto-kine secretion. M. avium subsp. paratuberculosis-specific lym-phocyte proliferation responses (LPRs) in blood were monitoredpreinfection in order to screen animals for prior exposure to M.avium subsp. paratuberculosis. Before infection, PBMCs from 1 of6 animals (calf 18) displayed a weakly positive LPR (SI � 2.8)(Table 1), and PBMCs from this animal failed to respond to invitro restimulation with the M. avium subsp. paratuberculosis ly-sate at 1 and 2 months postinfection. In vitro restimulation ofPBMCs with the M. avium subsp. paratuberculosis lysate atmonthly intervals following M. avium subsp. paratuberculosis in-fection induced strong LPRs (SI � 10) that were intermittent in50% (3/6) of the animals (Table 1). M. avium subsp. paratubercu-losis-specific LPRs were first detected with PBMCs at 2 monthspostinfection in 1 of 6 animals, and only 1 animal was consistentlypositive at all subsequent time points. At 9 months postinfection,4 of 6 animals had M. avium subsp. paratuberculosis-specific LPRsin mLNs associated with the intestinal segments, but M. aviumsubsp. paratuberculosis-specific LPRs were not detected in any an-imals when leukocytes isolated from the LP of infected compart-ments were assayed (Fig. 5). In contrast, stimulation with the M.avium subsp. paratuberculosis lysate resulted in significantly (P �0.05) greater TNF-� secretion by LPLs isolated from M. aviumsubsp. paratuberculosis-infected compartments than by LPLsfrom adjacent noninfected ileal compartments (Fig. 6A). The lev-els of M. avium subsp. paratuberculosis lysate-induced TNF-� se-cretion by PBMCs and mLN cells were not significantly greaterthan that observed for LPLs from the noninfected compartment.Similarly, stimulation of LPLs isolated from M. avium subsp.paratuberculosis-infected compartments and cells isolated fromdraining mLNs resulted in levels of IFN-� secretion significantly(P � 0.05) higher than those for LPLs isolated from noninfectedileal compartments. IFN-� secretion by both LPLs and mLN cells

FIG 4 TLR gene expression in M. avium subsp. paratuberculosis-infected andnoninfected ileal compartments. Expression of tlr-1 through tlr-10 in ilealtissues collected from M. avium subsp. paratuberculosis-infected and nonin-fected compartments (n � 3) was quantified by qRT-PCR. TLR expressionlevels are expressed as the change in the cycle threshold (CT) relative to the CT

of the housekeeping gene -actin. The CT was calculated by subtracting theCT value for -actin from the CT value for each TLR. The data presented aremeans and 1 SD for values from the three animals. Lower CT values representincreased expression levels for individual TLR genes.

TABLE 1 M. avium subsp. paratuberculosis-specific lymphocyteproliferation response monitored in PBMC

Calf

LPRa at the following mo postinfection:

0 1 2 3 4 5 6 7 9–11

16 � � � � �� 17 � � � � � � � � �18 � � � � �� 23 � � � � � � � � �25 � � �� 27 � � � � � � � � �a Symbols represent SIs as follows: �, �2.5; ��, �10; �, �2.5 (negative).

FIG 5 Comparison of M. avium subsp. paratuberculosis-specific LPRs inPBMCs, mLN cells, and LPLs. Cells were isolated from calves between 9 to11 months postinfection and were restimulated with the M. avium subsp.paratuberculosis lysate in vitro. Data are shown for cells isolated from mLNsdraining each intestinal segment, and LPLs were isolated from the M.avium subsp. paratuberculosis-infected compartment within each intesti-nal segment. M. avium subsp. paratuberculosis-specific LPRs (SI, �2.5)(dashed horizontal line) were observed only in mLNs (4/6) and PBMCs(2/6), not in LPLs.




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was significantly (P � 0.01) greater than that observed whenPBMCs were stimulated with the M. avium subsp. paratuberculosislysate. These observations indicated that M. avium subsp. paratu-berculosis-specific T cells were localized in the intestine at the siteof M. avium subsp. paratuberculosis infection. mLN T cell sub-populations proliferating in response to M. avium subsp. paratu-berculosis antigen were further characterized using flow cytom-etry. Cell proliferation analyses of mLN cells from animalsdisplaying M. avium subsp. paratuberculosis-specific LPRs (n � 4)(Fig. 5) revealed that CD4 cells (mean net proliferating popula-tion 1 standard deviation [SD], 4.2% 1.4%) and �� TcR Tcells (mean net proliferating population 1 SD, 3.6% 0.7%),but not CD8 T cells (mean net proliferating population 1 SD,0.2% 0.1%), displayed significant (P � 0.05) M. avium subsp.paratuberculosis-specific proliferation (Fig. 7).

DISCUSSION

The mechanisms by which M. avium subsp. paratuberculosisevades acquired immunity and establishes persistent infections inthe small intestine have been difficult to investigate because theintestinal mucosal immune system is dispersed over a large area,and isolation of mucosal immune cells is difficult. A variety ofmodel systems have been developed to study interactions betweenM. avium subsp. paratuberculosis and the bovine small intestine.Studies have characterized host-pathogen interactions when M.avium subsp. paratuberculosis infection is first established (8, 23),and more recently, models have been developed to monitor hostresponses during persistent infections (24, 25). With these mod-els, however, it was not possible to ensure that M. avium subsp.paratuberculosis infection remained localized at the initial site ofexposure, and M. avium subsp. paratuberculosis was shed in feces.By use of surgically isolated segments of the terminal small intes-tine and a dose and strain of M. avium subsp. paratuberculosissimilar to those used in previous studies, a persistent and localizedenteric infection was established when calves were infected be-tween the ages of 10 and 14 days. Although acid-fast organismswere rarely observed in tissue sections, PCR analysis confirmedthat infection persisted in the intestinal tissue and remained local-ized to the site of initial exposure (Fig. 2) for at least 9 to 11months.

A thickened intestinal wall and enlarged mLNs are character-istic gross pathological changes associated with clinical Johne’sdisease (37), and these lesions usually require 2 to 5 years to de-velop in cattle (38). In the current infection model, no gross in-

FIG 6 M. avium subsp. paratuberculosis-specific TNF-� and IFN-� secretionby LPLs, mLN cells, and PBMCs at 9 to 11 months postinfection. LPLs, mLNcells, and PBMCs (5 � 105 cells/well) were isolated at 9 to 11 months postin-fection and were restimulated with the M. avium subsp. paratuberculosis lysatein vitro for 48 h, and culture supernatants were assayed for TNF-� and IFN-�.(A) LPLs isolated from M. avium subsp. paratuberculosis-infected compart-ments secreted significantly higher levels of TNF-� (P � 0.05) than did LPLsfrom adjacent noninfected ileal compartments (Cntrl). (B) LPLs from M.avium subsp. paratuberculosis-infected compartments and cells isolated frommLNs draining each intestinal segment secreted significantly (P � 0.05) higherlevels of IFN-� than did LPLs isolated from noninfected ileal compartmentsand PBMCs. Data presented are means and 1 SD for values from 6 animals. *,P � 0.05; **, P � 0.01.

FIG 7 mLN T cell subpopulations proliferating in vitro following restimula-tion with the M. avium subsp. paratuberculosis lysate. mLN leukocytes werelabeled with PKH-26 prior to culture in the presence (MAP) or absence (me-dia) of the M. avium subsp. paratuberculosis lysate. Cultured leukocytes wererecovered after 72 h of culture and were labeled with MAbs to identify CD4,CD8, and ��TcR T cell subpopulations. Individual T cell subpopulations weregated and were analyzed for changes in PKH-26 fluorescence intensity usingModFit cell cycle analysis. The data presented are representative of results fromthe four animals displaying positive LPRs in Fig. 5. Cell cycle analysis revealedthat both CD4 and ��TcR T cells progressed through multiple cell cycles in thepresence of the M. avium subsp. paratuberculosis lysate.




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testinal abnormalities were apparent when M. avium subsp.paratuberculosis-infected compartments were compared to non-infected compartments at 9 to 11 months postinfection (Fig. 1A).Furthermore, dissemination of M. avium subsp. paratuberculosisto mesenteric lymph nodes is a common feature of natural Johne’sdisease (39) and has been reported following experimental infec-tion by either the oral route (40, 41) or injection into the intestinalwall (42). Thus, the absence of detectable M. avium subsp. para-tuberculosis in the mLNs at 9 to 11 months postinfection (Fig. 2) isnot consistent with previous reports for natural and experimentalinfections. The current observation may indicate that local muco-sal immune responses (Fig. 5) and the recruitment of effector cellsto the intestine (Fig. 3) effectively limited bacteria to the initial siteof inoculation within the isolated intestinal segment (Fig. 2). Al-ternatively, restricting M. avium subsp. paratuberculosis infectionto an isolated intestinal segment may reduce bacterial replicationand allow sufficient time for the host to mount an effective im-mune response.

Inflammatory infiltrates and granulomas in the LP with en-largement and shortening of the villi are characteristic pathologi-cal lesions described for M. avium subsp. paratuberculosis-infectedsheep (43) and cattle (37, 44). Similar pathological changes wereobserved in M. avium subsp. paratuberculosis-infected intestinalcompartments (Fig. 1C and D), and eosinophils frequently sur-rounded and infiltrated the cellular aggregates in the LP (Fig. 1D,arrows). Eosinophil infiltrates have been observed previously insome, but not all, models of M. avium subsp. paratuberculosisinfection (45, 46), and eosinophils have been reported to sur-round Mycobacterium tuberculosis granulomas (47, 48). It hasbeen speculated that eosinophils may enhance Mycobacterium bo-vis infections by inhibiting macrophage activation and providing asuitable microenvironment for mycobacterial growth (49).Therefore, the histological lesions observed at 9 to 11 monthspostinfection were consistent with a persistent M. avium subsp.paratuberculosis infection but with few detectable acid-fact bacilli.PCR analysis, however, confirmed that M. avium subsp. paratu-berculosis infection persisted in the intestinal tissues of all animalsat the initial site of infection but was absent from noninfectedintestinal compartments (Fig. 2). The absence of M. avium subsp.paratuberculosis in noninfected compartments also indicated thatcalves were not exposed to M. avium subsp. paratuberculosis priorto experimental infection. Therefore, the current infection modelmay be very useful for screening vaccine candidates to determineif immunization can effectively clear M. avium subsp. paratuber-culosis infection following exposure to an infectious dose that es-tablishes a persistent infection in all animals.

Macrophages and T cells are key populations in the establish-ment and control of mycobacterial infections, and both of thesecell types accumulated in the LP following M. avium subsp. para-tuberculosis infection (44). Macrophages form granulomas shortlyafter infection, and during the first 2 to 4 weeks postinfection, Tcells also surround the nascent granuloma (50). Our infectionmodel provided an opportunity to quantify and further character-ize the cellular changes occurring in the LP during a persistent M.avium subsp. paratuberculosis infection. This is the first evidencethat persistent M. avium subsp. paratuberculosis infection is asso-ciated with an accumulation of DCs as well as macrophages in theLP (Fig. 3A). The selective recruitment of myeloid cells during M.avium subsp. paratuberculosis infection has been attributed to in-creased expression of cytokines and chemokines (8, 51–53). The

specificity of this recruitment during M. avium subsp. paratuber-culosis infection is supported by the comparison between infectedand noninfected compartments (Fig. 3A). Increased DC recruit-ment may have significant implications for the processing andpresentation of mycobacterial antigens (54). DCs are the only an-tigen-presenting cells with migratory properties, and they con-tribute to the induction of immune responses to mycobacteriaboth locally (54) and in the draining lymph node (55). The induc-tion of M. avium subsp. paratuberculosis-specific T cell responsesin the draining mLN (Fig. 5 and 6) in the absence of detectableacid-fast bacteria may be consistent with the delivery of M. aviumsubsp. paratuberculosis antigens to this immune induction site byDCs. Furthermore, the identification of tolerogenic mucosal DCsin the small intestine (reviewed in references 31 and 56) raises thepossibility that the outcome of M. avium subsp. paratuberculosisinfection may be influenced by the type of DCs recruited to thesmall intestine and acquiring M. avium subsp. paratuberculosisantigens. The present observations suggest that further investiga-tion of the interactions between M. avium subsp. paratuberculosisand mucosal DC subpopulations may be important for full under-standing of the pathogenesis of Johne’s disease.

A key feature of the immune response to M. avium subsp.paratuberculosis is a strong cell-mediated immune response atearly stages of infection and during subclinical infection (57).Analysis of T cells in the LP revealed a significant (P � 0.05)accumulation of CD8� and ��TcR cells at the site of M. aviumsubsp. paratuberculosis infection (Fig. 3B). The increased frequen-cies of both CD8� and ��TcR cells in M. avium subsp. paratuber-culosis-infected ileal compartments may be consistent with thecoexpression of CD8 on bovine ��TcR effector cells (58) and areconsistent with previous reports that ��TcR and CD8� T cellnumbers increase in the laminae propriae of cows and goats withclinical Johne’s disease (59, 60). Although ��TcR cells have beenlinked to protection against intracellular pathogens (61), an invitro model of M. avium subsp. paratuberculosis infection revealedthat ��TcR cells did not enhance the bactericidal function of M.avium subsp. paratuberculosis-infected macrophages (62). Thesein vitro experiments were limited by the use of ��TcR cells isolatedfrom lymph nodes and restimulation of ��TcR cells with purifiedprotein derivative (PPD). Although CD4 and ��TcR T cells iso-lated from the mLN could be restimulated in vitro with soluble M.avium subsp. paratuberculosis proteins (Fig. 7), the phenotype of Tcells secreting TNF-� and IFN-� in LPLs (Fig. 6) was not deter-mined. The current results (Fig. 5 and 6) indicate that distinct M.avium subsp. paratuberculosis-specific effector T cell subpopula-tions are present in different mucosal immune compartments.This may have important implications for the screening of poten-tial vaccine candidates. The mLN functions as the major site forthe induction of mucosal immune responses and may provide anappropriate source of T cells for screening of individual M. aviumsubsp. paratuberculosis proteins to identify those recognized dur-ing infection (Fig. 6). The use of cytokine secretion assays may bemore appropriate, however, for the screening of antigens to deter-mine which M. avium subsp. paratuberculosis proteins are recog-nized by effector cells localized in the LP at the site of M. aviumsubsp. paratuberculosis infection in the intestine.

LPL populations involved in innate and acquired immune re-sponses were modified following persistent M. avium subsp. para-tuberculosis infection (Fig. 3). The efficiency with which the im-mune system recognizes invading pathogens is influenced by




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germ line-encoded pattern recognition receptors (PRR), and Toll-like receptors (TLRs) are one of the most studied families of PRR(63). Mycobacterial components are recognized by TLR2, TLR2/TLR1 (TLR2/1), and TLR2/6 dimers and by TLR4 (64–67). Alter-ations in TLR6 (65), TLR1 to -5, and TLR8 gene expression (66)have been associated with Johne’s disease in cattle and sheep.Therefore, it was expected that TLR gene expression in the intes-tine would be significantly altered following M. avium subsp.paratuberculosis infection, especially when myeloid cell popula-tions were significantly altered (Fig. 3A). Further studies will berequired to determine whether the present observations simplyreflect the low level of M. avium subsp. paratuberculosis infectionor whether analysis of TLR expression in purified myeloid sub-populations is required to reveal specific alterations in myeloidcell function during M. avium subsp. paratuberculosis infection inthis model system.

The current results support the conclusion that surgically iso-lated bovine ileal segments provide an effective model system foranalyzing local mucosal immune responses during a persistent M.avium subsp. paratuberculosis infection. The intestinal segmentmodel restricted M. avium subsp. paratuberculosis infection to theinitial site of delivery (Fig. 2), ensured that a consistent dose ofbacteria was delivered to all animals, and provided an internalcontrol for defining M. avium subsp. paratuberculosis-specificmucosal immune responses (Fig. 3, 5, and 6). Analysis of effectorT cell antigen specificity in mucosal immune compartments andthe study of interactions between M. avium subsp. paratuberculo-sis and mucosal DCs will be critical for the rational design of avaccine to prevent M. avium subsp. paratuberculosis infection andthe shedding of bacteria by infected animals.

ACKNOWLEDGMENTS

This research was funded by the Saskatchewan Agriculture DevelopmentFund, the Beef Cattle Research Council, and the Natural Sciences andEngineering Research Council of Canada. Philip Griebel is supported by aTier I CRC in Neonatal Mucosal Immunology, which is funded by theCanadian Institutes of Health Research.

We gratefully acknowledge the VIDO Animal Care staff for assistancein veterinary care, animal handling, and sample collection. We also ac-knowledge Donna Dent, who performed the cytokine ELISAs, for techni-cal support; Kelli Bell for assistance with bacterial culture; and Yurij Po-powych and Natasa Arsic for assistance with cell isolation and FACSanalysis.

We declare that we have no conflicts of interest.

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