What Do Peyer's Patches in Immunosuppressed Juvenile Animals Look Like
Infect Immun. 2010 Aug; 78(8): 3570–3577.
Peyer's Patch-Deficient Mice Demonstrate That Mycobacterium avium subsp. paratuberculosis Translocates across the Mucosal Barrier via both M Cells and Enterocytes only Has Inefficient Dissemination ▿
Luiz E. Bermudez
Kuzell Establish, California Pacific Medical Centre Inquiry Institute, San Francisco, California 94115,1 School of Veterinary Medicine and Biomedical Sciences, Academy of Nebraska, Lincoln, Nebraska 68583-0905,2 Department of Biomedical Sciences, College of Veterinarian Medicine,3 Section of Microbiology, College of Science, Oregon State University, Corvallis, Oregon 97331iv
Mary Petrofsky
Kuzell Institute, California Pacific Medical Center Research Institute, San Francisco, California 94115,1 School of Veterinary Medicine and Biomedical Sciences, University of Nebraska, Lincoln, Nebraska 68583-0905,2 Department of Biomedical Sciences, College of Veterinarian Medicine,3 Section of Microbiology, College of Science, Oregon Country University, Corvallis, Oregon 97331iv
Sandra Sommer
Kuzell Establish, California Pacific Medical Center Research Institute, San Francisco, California 94115,ane School of Veterinary Medicine and Biomedical Sciences, University of Nebraska, Lincoln, Nebraska 68583-0905,two Section of Biomedical Sciences, College of Veterinary Medicine,3 Section of Microbiology, College of Science, Oregon State University, Corvallis, Oregon 973314
Raúl K. Barletta
Kuzell Plant, California Pacific Medical Center Research Institute, San Francisco, California 94115,one School of Veterinary Medicine and Biomedical Sciences, Academy of Nebraska, Lincoln, Nebraska 68583-0905,2 Department of Biomedical Sciences, College of Veterinary Medicine,3 Section of Microbiology, Higher of Science, Oregon Country Academy, Corvallis, Oregon 973314
Received 2009 Dec 17; Revised 2010 Feb five; Accepted 2010 May fourteen.
Abstruse
Mycobacterium avium subsp. paratuberculosis, the amanuensis of Johne's affliction, infects ruminant hosts by translocation through the intestinal mucosa. A number of studies take suggested that M. avium subsp. paratuberculosis interacts with Thousand cells in the Peyer'southward patches of the small intestine. The invasion of the intestinal mucosa by K. avium subsp. paratuberculosis and Mycobacterium avium subsp. hominissuis, a pathogen known to interact with abdominal cells, was compared. M. avium subsp. paratuberculosis was capable of invading the mucosa, but it was significantly less efficient at dissemination than M. avium subsp. hominissuis. B-cell knockout (KO) mice, which lack Peyer's patches, were used to demonstrate that M. avium subsp. paratuberculosis enters the intestinal mucosa through enterocytes in the absenteeism of M cells. In improver, the results indicated that M. avium subsp. paratuberculosis had equal abilities to cross the mucosa in both Peyer's patch and non-Peyer'due south patch segments of normal mice. Grand. avium subsp. paratuberculosis was also shown to interact with epithelial cells past an α5βi integrin-contained pathway. Upon translocation, dendritic cells ingest One thousand. avium subsp. paratuberculosis, simply this process does not lead to efficient dissemination of the infection. In summary, One thousand. avium subsp. paratuberculosis interacts with the abdominal mucosa by crossing both Peyer's patches and not-Peyer'south patch areas merely does non translocate or disseminate efficiently.
Mycobacterium avium subsp. paratuberculosis is the agent of Johne'southward disease, a debilitating condition of cattle and other ruminants that is associated with severe diarrhea and wasting (10, 32). Johne'south disease causes significant economic loss, with a severe impact on the dairy manufacture (5, 37). Some studies take implicated M. avium subsp. paratuberculosis as i of the potential etiologic agents or opportunistic pathogens of Crohn's illness patients (5, 37).
Thousand. avium subsp. paratuberculosis infects immature calves and is ordinarily transmitted by contaminated stools or milk (10, 29). In the first steps of pathogenesis, the bacterium crosses the intestinal mucosa of the infected host. Previous work has suggested that M. avium subsp. paratuberculosis crosses the intestinal mucosa by entering Yard cells in the Peyer's patches of calves (15). More recently, a goat kid animal model suggested that the ports of entry in the intestinal mucosa are non limited to M cells (24). In addition, work in vitro showed that M. avium subsp. paratuberculosis can invade bovine kidney epithelial (17) and murine intestinal epithelial (xx) cells efficiently. Mycobacterium avium subsp. hominissuis, a subspecies related to M. avium subsp. paratuberculosis (26), invades the intestinal mucosa by interacting primarily with enterocytes (4, 21). AIDS patients acquire G. avium subsp. hominissuis infections mainly through the gastrointestinal tract (6), and studies of macaques take demonstrated that this microorganism is inside enterocytes (L. E. Bermudez et al., unpublished observations). Information technology should be noted that in before literature, this subspecies is referred to merely every bit Mycobacterium avium or Mycobacterium avium subsp. avium (five, 12, 31); the new taxonomic description (M. avium subsp. hominissuis) for microorganisms isolated from humans and swine is relatively recent (14) and was non immediately captured or accepted past the scientific community at big (31). Still, in spite of the close genetic relationship, M. avium subsp. hominissuis is likely the only truly ecology mycobacterium (33), oft acquired by humans and pigs, only rarely isolated from birds or cattle (33). In contrast, M. avium subsp. paratuberculosis is the predominant, or perhaps the just, mycobacterium isolated from ruminants with Johne's disease and associated with cases of Crohn's disease in humans, but rarely isolated from AIDS patients. Thus, these subspecies correspond microorganisms associated with unique disease entities and epidemiological distributions.
The mouse model of M. avium subsp. paratuberculosis may not entirely reverberate the affliction in cattle, sheep, and goats; nevertheless, it has value for studying aspects of M. avium subsp. paratuberculosis pathogenesis in small laboratory animals (xi, 16). M. avium subsp. paratuberculosis has been observed multiplying in the intestinal mucosa of athymic nude gnotobiotic mice (9), indicating that the bacterium can replicate efficiently in the intestinal mucosa in the absence of a competent immune system. The availability of recombinant mouse strains offers the opportunity to accost the roles of M cells and enterocytes in the translocation of M. avium subsp. paratuberculosis through the intestinal wall. To study this process, we compared M. avium subsp. paratuberculosis dissemination in wild-type mice and B-cell knockout (KO) mice, which lack Peyer's patches and M cells. Our results point that Grand. avium subsp. paratuberculosis crosses the intestinal mucosa by invading both Grand cells and enterocytes and that it tin can translocate, although with less efficiency than M. avium subsp. hominissuis, beyond mucosal epithelial cells. Leaner likewise infected dendritic cells after crossing the epithelial barrier.
MATERIALS AND METHODS
Bacteria.
1000. avium subsp. paratuberculosis strain K-10 is a bovine isolate capable of causing disease in both cattle and mice (xviii). M. avium subsp. paratuberculosis was grown on Middlebrook 7H10 agar plates supplemented with 2 mg/liter of mycobactin J and oleic acrid, albumin, dextrose, and catalase (OADC). Grand. avium subsp. hominissuis strain 101 is capable of infecting immunocompetent and immunosuppressed mice (2). G. avium subsp. hominissuis was cultured on 7H10 agar supplemented with OADC. Bacteria grown for 14 days (Grand. avium subsp. hominissuis) or 20 days (M. avium subsp. paratuberculosis) were used in the experiments. Staphylococcus aureus SA101 is a clinical isolate obtained from the purulent material of a cutaneous abscess. For the experiments, this bacterium was cultured on Mueller-Hilton agar plates for 48 h.
Mice.
Eight- to 10-calendar week-old female pathogen-free C57BL/6J black mice, weighing 25 thousand, were obtained from the Jackson Laboratory (Bar Harbor, ME) and were used later on 2 weeks of quarantine. C57BL/6J B-prison cell-deficient mice (immunoglobulin H6 negative, 8 to 10 weeks old, weighing 25 g) were purchased from the Jackson Laboratory and were used later on two weeks of quarantine. These animals accept previously been shown to lack Peyer'south patches and M cells (8). In some experiments, 10- to 12-calendar week-old mice were used. All experiments were performed co-ordinate to the guidelines of the institutional animal intendance and utilise committee.
Infection of mice.
The first set of experiments compared the infection of C57BL/6J mice by Yard. avium subsp. paratuberculosis versus M. avium subsp. hominissuis. Bacteria were cultured on solid medium as described above, with the inoculum prepared at 2.4 × 10seven CFU/ml (Thou. avium subsp. hominissuis 101) or iii.8 × 107 CFU/ml (Thousand. avium subsp. paratuberculosis K-x). Mice were given 0.1 ml of the bacterial suspension per os and were followed for i, 2, 4, 8, and sixteen weeks postinfection (wpi). Ten mice were used for each time bespeak in each of 2 sets of experiments. At each time point, mice were killed, and the spleens, livers, and terminal ilea were harvested, homogenized, serially diluted, and plated onto Middlebrook 7H10 agar plates supplemented with OADC, mycobactin J, and PACT (polymyxin B at 5 μg/ml, amphotericin B at iv.5 μg/ml, carbenicillin at 22 μg/ml, and trimethoprim at 2 μg/ml).
Intestinal loop.
The intestinal-loop assay was carried out basically as described previously for M. avium subsp. hominissuis (21). Briefly, both C57BL/6 blackness mice and C57BL/6J B-cell-deficient mice (immunoglobulin H6 negative), viii to 10 weeks old, weighing 25 g, were anesthetized by intraperitoneal administration of phenobarbital. Mice were maintained nether profound anesthesia during the entire procedure. Post-obit anesthesia, the abdominal cavity was carefully opened, and a segment of the small intestine that was approximately iii cm long, proximal to the ileocecal expanse, was identified. A suture line was tied at both ends of the segment of intestine, tightly enough to close the intestinal lumen while not interfering with the claret flow. A interruption containing approximately 1 × 107 Chiliad. avium subsp. hominissuis or ii.5 × ten7 M. avium subsp. paratuberculosis organisms in Hanks' balanced salt solution (HBSS) was injected into the proximal position of the isolated intestinal segment. Mice were kept alive for ane and iii h, after which they were killed, and the intestinal segment was removed, opened longitudinally, and rinsed with HBSS to remove unbound or weakly bound leaner. The removed intestinal segment was placed in v ml of 7H9 broth with xxx% glycerol and was homogenized. The suspension was then serially diluted in 7H9 goop before beingness plated onto 7H10 agar, supplemented with mycobactin J and PACT to inhibit intestinal biota, for quantification of feasible organisms associated with the intestinal mucosa-submucosa. Plates were cultured for several days at 37°C, and the concentration of leaner (expressed as CFU per gram of tissue) was calculated as (average CFU per plate × dilution factor × 5 ml)/(intestinal-segment weight).
Differential uptake by Peyer's patch and non-Peyer's patch segments.
To determine whether Chiliad. avium subsp. paratuberculosis enters the intestinal mucosa preferentially by Thou cells or enterocytes, we performed an in vivo assay in which x-calendar week-old C57BL/6J mice were given K. avium subsp. paratuberculosis (3 × 10viii bacteria) orally. At 4 h and 24 h, the mice (5 mice per time point per experimental group) were sacrificed. Their abdomens were opened, and for each mouse, five segments (length, 1 cm) comprising regions with Peyer's patches and 5 segments without Peyer'south patches were obtained, opened longitudinally, done, homogenized, and plated onto 7H10 agar with mycobactin J.
Histopathology.
The abdominal ilea from four wild-type mice and four B-cell KO mice were obtained, fixed with 10% neutral buffered formalin, and stained with hematoxylin-eosin or an acrid-fast stain equally previously described (13).
Electron microscopy.
Mice were infected with K. avium subsp. paratuberculosis, and i or ii days after infection, the animals were harvested; the intestinal segment was cutting and opened longitudinally, briefly washed in HBSS, and immersed in iv% paraformaldehyde and ii% glutaraldehyde for 12 h at 4°C. And so the material was placed in HBSS and stained with a i% aqueous solution of osmium tetroxide for i h at 4°C, as previously described (17). Samples were dehydrated in ethanol at room temperature, embedded in resin, and polymerized at 52°C. Photographs were taken using a transmission electron microscope (EM) at a magnification of ×ten,000.
Translocation analysis.
Translocation assays were performed equally previously described (17), using the Transwell 2-sleeping room civilisation system (Costar; Corning, NY) containing a 0.33-cm2 porous membrane (pore size, iii.0 μm). Monolayers were established on the top of the membrane by seeding it with 1 × 10half-dozen Madin-Darby bovine kidney (MDBK) cells in Dulbecco's modified Hawkeye's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). The culture medium was changed every 2 days, and the integrity of the monolayer was monitored past the following methods: (i) microscopic ascertainment, (2) measurement of the transwell resistance using a Millipore (Bedford, MA) transwell device equally reported elsewhere (17), and (3) the trypan blue (0.25%) permeability analysis (optical density at 580 nm), as described previously (iii). Trypan blueish (0.25%) was added to the monolayer, and iii h afterward, the supernatant of the lesser chamber was obtained for a spectrophotometer reading. The control included the medium alone (baseline). The peak sleeping accommodation was infected either with 5 × 10v bacteria or with 2 × 10five bacteria that had previously been exposed to whole cow milk, as described previously (17). Afterward 2, 6, or 24 h of infection, 500 μl of filtrate was collected from the bottom chamber, and the system was replenished with fresh culture medium. The translocation ability was calculated as the cumulative percent of the initial inoculum that was recovered in the lesser chamber at each time point.
Bacterial uptake assays.
Bacteria (ten5) were added to MDBK cells in medium with 10% FBS for 1 h. Extracellular leaner were removed, and monolayers were washed three times with HBSS. Monolayers were then lysed and the number of intracellular bacteria quantified.
Bovine dendritic cells.
Bovine dendritic cells were obtained by differentiation of bovine monocytes by treatment with human recombinant granulocyte-macrophage colony-stimulating cistron (GM-CSF; x μg/ml) and interleukin-4 (IL-iv; ten μg/ml) (Genzyme, Cambridge, MA). Mononuclear phagocytes from healthy cows at the Oregon State Academy (OSU) dairy subcontract, which are negative for Johne's disease, were isolated by using Percoll density gradient. Monocytes were then resuspended in RPMI 1640 supplemented with ten% FBS. The monocytes obtained were seeded at two × 10five per well and were treated with recombinant cytokines. Monocytes were matured for 7 days (35, 36) and were used when the morphology was indicative of dendritic cells. Dendritic cells were and then maintained in RPMI 1640 supplemented with x% FBS. To infect the dendritic cells (approximately x5 cells), nosotros used 10half dozen bacteria for 1 h. After, monolayers were washed 3 times, and the lysate was plated onto 7H10 agar with mycobactin J. In some experiments, polarized monolayers of MDBK cells in a Transwell system were infected with 5 × 106 M. avium subsp. paratuberculosis leaner for 2 h. Twenty-4 hours subsequently the infection, the translocated bacteria in the supernatant in the lower chamber were quantified. The jail cell culture supernatant was obtained and transferred to a 24-well plate with adherent dendritic cells. Then i × x5 dendritic cells were incubated with 1 × 105 translocated M. avium subsp. paratuberculosis bacteria for one h. As controls, bacteria in 7H9 medium supplemented with mycobactin J were used. Monolayers were repeatedly washed and lysed, and the internalized leaner were quantified.
Statistical analysis.
The comparisons among experimental groups and the command were evaluated for statistical significance by using Student's t test or the Mann-Whitney examination.
RESULTS
Infection of C57BL/6J with M. avium subsp. hominissuis or Thousand. avium subsp. paratuberculosis.
To evaluate whether M. avium subsp. paratuberculosis invades the intestinal mucosae of mice, and if information technology does then differently from Grand. avium subsp. hominissuis, mice were infected orally, and the numbers of leaner in the terminal ileum, liver, and spleen were monitored over time. In C57BL/vi black mice, the abdominal bacillary counts for M. avium subsp. paratuberculosis increased monotonically during the 16-calendar week period. In contrast, Chiliad. avium subsp. paratuberculosis did not grow to pregnant numbers in the liver or spleen at 4 wpi, but moderate dissemination to the liver and the spleen was observed at 8 and sixteen wpi (Fig. 1 A). Intestines remained the organ with the greatest microbial burden during this period. Infection with M. avium subsp. hominissuis followed a different class, with bacillary counts increasing monotonically in the liver and the spleen, while intestinal counts remained approximately constant, increasing only at xvi wpi (Fig. 1B ). Comparison on a per-organ basis led to the observation that 1000. avium subsp. paratuberculosis and M. avium subsp. hominissuis reached statistically significantly different levels in the intestines of infected animals, but the difference was less than 100-fold for all cases (Fig. 1C ). Dramatic differences, with extremely loftier counts for Thou. avium subsp. hominissuis-infected animals, were observed in the liver (Fig. 1D ) and the spleen (Fig. 1E ). In all cases, Grand. avium subsp. hominissuis burdens 500-fold greater than 1000. avium subsp. paratuberculosis burdens were observed. In summary, M. avium subsp. paratuberculosis is less efficient at dissemination in the host than M. avium subsp. hominissuis. These results further confirmed that the mouse tin can exist used equally a model for investigating the interaction between M. avium subsp. paratuberculosis and the intestinal mucosa.
Microbial burdens in organs of infected C57BL/6 black mice. (A and B) Mice were infected equally described in Materials and Methods, and microbial burdens in animals infected with M. avium subsp. paratuberculosis (MAP) (A) or M. avium subsp. hominissuis (MAH) (B) were adamant as a function of time. Note that unlike scales are used for the y axes in panels A and B. (C, D, and E) The same information are plotted by using the same y axis scale for all graphs, highlighting the comparison of organ burdens betwixt the two M. avium subspecies. Mice were infected with two.4 × 10half-dozen One thousand. avium subsp. hominissuis organisms or with three.8 × 106 Thou. avium subsp. paratuberculosis organisms. Each bar represents the hateful data collected from 20 mice. Error confined, standard errors of the means. P < 0.05 for the comparison between Chiliad. avium subsp. hominissuis and M. avium subsp. paratuberculosis at the same time point for each of the organs.
Intestinal loop.
To make up one's mind whether M. avium subsp. paratuberculosis needs Peyer'due south patches in lodge to cross the intestinal mucosa, we used an intestinal-loop model with C57BL/6 wild-blazon and B-jail cell knockout mice, which lack Peyer's patches. Equally seen in Table one , both Thousand. avium subsp. paratuberculosis and M. avium subsp. hominissuis (used as a control) (21) are able to enter the abdominal mucosa every bit well in the presence or absenteeism of Peyer's patches. That finding, though, does not necessarily point that M. avium subsp. paratuberculosis invades the abdominal mucosa through the Peyer's patches and Peyer's patch-deficient segments in the wild-type mouse.
Tabular array 1.
Efficacy of invasion of the intestinal mucosae of C57BL/6 mice and B-prison cell-deficient mice (defective Peyer's patches) by M. avium subsp. paratuberculosis using an intestinal-loop model a
| Bacterium | % of inoculum inside the mucosa b | |||
|---|---|---|---|---|
| C57BL/6J mice | B-cell-deficient mice | |||
| 1 h | iii h | 1 h | iii h | |
| Thousand. avium subsp. paratuberculosis | 5.one ± 0.three | 36 ± x | four.3 ± 0.five | 30 ± half-dozen |
| M. avium subsp. hominissuis | 7.six ± 0.4 | 54 ± 9 | vi.eight ± 0.iii | 52 ± iv |
Preferential site of invasion.
To determine the site of entry of M. avium subsp. paratuberculosis into the intestinal tract, mice were infected orally, and 3 segments (1 cm each) containing Peyer's patches and three segments containing non-Peyer's patch regions were homogenized and plated. As shown in Table 2 , M. avium subsp. paratuberculosis enters equally well into the mucosal area of Peyer's patches and into surrounding areas that do not incorporate M cells. Moreover, Grand. avium subsp. paratuberculosis bacilli were visualized in association with enterocytes by transmission electron microscopy (Fig. 2 ).
Transmission electron micrograph of a sample from a mouse orally infected with M. avium subsp. paratuberculosis. The intestine was harvested at 2 days postinfection. M. avium subsp. paratuberculosis can be observed inside enterocytes (arrows). Magnification, ×10,000.
TABLE two.
Preferential site of Chiliad. avium subsp. paratuberculosis invasion of the abdominal mucosae of C57BL/6J mice
| Time | Concn of leaner (CFU/k of intestinal tissue) a | |
|---|---|---|
| Peyer's patch segments | Non-Peyer's patch segments | |
| 4 h | 3.6 ± 0.4 | 4.1 ± 0.8 |
| 24 h | viii ± ii | nine.3 ± 1.7 |
To determine whether the enterocyte road was important for infection, we carried out an in vivo experiment in which wild-blazon C57BL/6J mice were infected orally with Chiliad. avium subsp. paratuberculosis, and 4 h and 24 h later, the number of leaner in the intestinal wall was determined. As shown in Table two , comparison between the number of bacteria in Peyer'south patch and non-Peyer'southward patch areas identified a slight preference for the leaner to enter the non-Peyer's patch segments, though the differences were not statistically significant (P > 0.05). Histopathological observations indicated that wild-type mice infected with M. avium subsp. paratuberculosis displayed approximately the same number of bacteria in the submucosa as that in corresponding tissues from B-cell KO mice (data not shown).
Interactions with a polarized monolayer.
M. avium subsp. paratuberculosis incubated with polarized MDBK epithelial cells crossed the monolayer from the upmost to the basolateral surface. As shown in Table 3 , significant crossing occurs later two h. Crossing of the monolayer by M. avium subsp. paratuberculosis did non alter the transmembrane resistance. When bacteria were incubated in the presence of whole milk, a significantly greater number of organisms translocated MDBK cells at early fourth dimension points (Table 3 ).
TABLE 3.
Translocation of Grand. avium subsp. paratuberculosis across an epithelial cell monolayer
| Status | two h | 6 h | 24 h | |||
|---|---|---|---|---|---|---|
| % of inoculum that translocated | Transmembrane resistance (Ω) a | % of inoculum that translocated | Transmembrane resistance (Ω) | % of inoculum that translocated | Transmembrane resistance (Ω) | |
| No exposure to milk (v × tenfive bacteria) | 0.2 ± 0.05 | 320 ± 10 | 0.4 ± 0.04 c | 310 ± xxx | 0.6 ± 0.05 | 330 ± 24 |
| Preincubation with milk (2 × x5 leaner) b | 0.four ± 0.06 | 320 ± 26 | 0.7 ± 0.04 c , d | 328 ± 24 | 1.v ± 0.06 c , d | 325 ± 39 |
Role of αfiveβane integrin in M. avium subsp. paratuberculosis uptake by MDBK cells.
By work had suggested the possible role of fibronectin in the translocation of the bacterium through the intestinal tract. To investigate if M. avium subsp. paratuberculosis invades MDBK cells using the fibronectin receptor, we treated MDBK monolayers with a mouse anti-α5βi IgG antibody or with an irrelevant anti-Escherichia coli lipopolysaccharide (LPS) IgG2a antibody at 30 μg/ml. Because we did not know whether the anti-human integrin would bind to the bovine antigen, we used a Staphylococcus aureus clinical isolate as a control. Incubation of S. aureus in the presence of the anti-α5βane antibiotic led to an 85% ± 5% reduction in uptake in 1 h. In contrast, the entry of G. avium subsp. paratuberculosis was reduced past 37.iii%. In the initial inoculum, most of the M. avium subsp. paratuberculosis, which was internalized past MDBK cells, entered by a different pathway than the fibronectin receptor. Every bit shown in Table 4 , the command antibody results support the absenteeism of nonspecific furnishings.
Table iv.
Touch on of the β1 integrin receptor on the entry of M. avium subsp. paratuberculosis into MDBK cells
| Experimental grouping | South. aureus | M. avium subsp. paratuberculosis | ||
|---|---|---|---|---|
| No. of intracellular bacteria a | % Δ b | No. of intracellular leaner a | % Δ | |
| Control (no antibody) | (3.iv ± 0.5) × 104 | (2.6 ± 0.4) × 103 | ||
| Anti-α5βane | (5.1 ± 0.3) × 103 | 85 c | (9.seven ± 0.2) × ten2 | 37.3 d |
| Anti-LPS | (3.2 ± 0.four) × 104 | −v d | (3.0 ± 0.3) × 103 | 15 d |
Uptake by dendritic cells.
M. avium subsp. paratuberculosis was placed in the presence of dendritic cells, and the percentage of leaner that were internalized was adamant. After xv min, (iii ± 0.four) × 101 M. avium subsp. paratuberculosis bacteria were observed inside dendritic cells (0.003% of the inoculum), while at thirty min and 1 h, (6 ± 0.vi) × 102 (0.01% of the inoculum) and (ane.5 ± 0.3) × 104 (1.8% of the inoculum) bacteria were ingested past dendritic cells, respectively (Table 5 ). Since G. avium subsp. paratuberculosis is likely to meet dendritic cells upon translocation of the mucosal epithelium, we infected polarized MDBK cells and used the translocated bacteria to infect dendritic cells. As shown in Fig. 3 , translocated bacteria were significantly more efficiently internalized by dendritic cells than bacteria grown on plates. Observation of monolayers for as long as 10 days showed a stable number of intracellular bacteria without significant growth.
Uptake and survival of M. avium subsp. paratuberculosis in dendritic cells. (A) Uptake of 1000. avium subsp. paratuberculosis past bovine dendritic cells post-obit translocation beyond a polarized monolayer of MDBK cells. Uptake by dendritic cells was immune for 1 h. Bacteria obtained from 7H9 goop medium supplemented with mycobactin J were used as a command. (B) Number of M. avium subsp. paratuberculosis bacteria in dendritic cells x days afterward infection (no significant differences in growth were observed).
TABLE 5.
Infection of bovine dendritic cells a by Chiliad. avium subsp. paratuberculosis
| Time | No. of intracellular bacteria | % of inoculum |
|---|---|---|
| Uptake | ||
| 15 min | (3 ± 0.4) × 101 | 0.003 |
| xxx min | (6 ± 0.6) × tenii | 0.01 |
| Intracellular growth | ||
| 1 h | (1.5 ± 0.iii) × 104 | |
| four days | (iii.8 ± 0.3) × 104 | 1.8 |
Word
In this study, we developed a novel low-dose (ca ii.v × xseven-CFU) oral-infection model of mice in social club to compare the pathogenesis of M. avium subsp. hominissuis and Chiliad. avium subsp. paratuberculosis. In dissimilarity, other studies using oral infection of mice have used a significantly higher dose (1.0 × 10x to i.0 × 1011 CFU). High-dose oral experimental infection with G. avium subsp. paratuberculosis resembles the intraperitoneal infection model, resulting in higher microbial burdens in the livers and spleens of infected animals than in the intestines (eleven). Our model reflects more than closely the natural infection of ruminants, in that at that place is a higher level of colonization of the abdominal mucosa by One thousand. avium subsp. paratuberculosis early in the infection (up to 4 wpi). Notwithstanding, at a similar infection dose, M. avium subsp. hominissuis is significantly more effective at disseminating in the host, reaching loftier colonization levels in the liver and spleen. Thus, this model reveals significant differences in the pathogenesis of these subspecies, as reflected in natural infections. In this context, G. avium subsp. paratuberculosis infects calves, and most of the bacteria remain in the intestinal wall, leading to lesions feature of Johne's diseases in the developed beast (25). A pocket-size percentage of the leaner disseminate, with some spreading to distant tissues, such every bit the mammary gland (27) and local lymph nodes (28). M. avium subsp. hominissuis, in contrast, crosses the intestinal mucosa by entering enterocytes (21), where it suppresses chemokine production (22) and, therefore, inflammatory cell migration (xiii). M. avium subsp. hominissuis disseminates to mesenteric lymph nodes (xix) and, in immunosuppressed individuals, to afar sites such equally spleen and bone marrow (12).
K. avium subsp. paratuberculosis infects young calves through the gastrointestinal tract (9, 12, 27). Information technology has been hypothesized that this microorganism crosses the intestinal mucosa by the Peyer's patches, reaching the submucosa (12, xix). Still, a recent study with goat kids suggested that 1000. avium subsp. paratuberculosis enters the intestinal mucosa, translocating through enterocytes (20). Past using B-jail cell knockout mice deficient in Peyer's patches, it was observed that M. avium subsp. paratuberculosis can even so enter the abdominal wall. Chiliad. avium subsp. paratuberculosis can infect the mucosa similarly in C57BL/6J wild-type mice and in B-jail cell knockout animals, and when given orally, the bacterium can be constitute both in regions with Peyer'south patches and in regions in which there are no Peyer's patches. The histopathological alterations at the initial phase of infection appear to be the aforementioned, suggesting that the two subspecies cross the mucosa similarly, merely the kinetics of dissemination differ significantly. The molecular basis for this difference betwixt these closely related organisms is unknown. Nonetheless, comparison of the two genomes unveiled the occurrence of genome segments specific for either G. avium subsp. paratuberculosis or M. avium subsp. hominissuis (38). The study of these genomic regions might assistance to explain these phenotypic differences. Alternatively, differential gene expression of primal virulence determinants in these two subspecies may business relationship for these observations.
By using a mouse strain lacking Peyer's patches (B-cell KO mice) and comparing the level of infection with that of the wild-type mouse, it became evident that the levels of tissue infection were similar, independently of the existence of Peyer's patches. In the presence of Peyer's patches, the bacterium used the Thou cells to cantankerous the mucosa, but in the absence of the patches, M. avium subsp. paratuberculosis was capable of interacting with enterocytes (Fig. 2 ). Additional experiments confirmed that bacteria delivered orally infected both Peyer's patches and non-Peyer's patch regions (Fig. iv depicts a schematic model). Whether the mechanisms of interaction are the same is currently unknown. Bacterial genes required for the invasion of bovine epithelial cells have been identified (one), merely all the evidence then far supports the hypothesis that uptake by M cells follows binding to the βi integrin receptor (fifteen, 23, 24). Our study confirmed that in vitro, invasion of MDBK cells by M. avium subsp. paratuberculosis does not depend on the β1 integrin receptor as a primary mechanism of uptake.
Mouse model of M. avium infection. Shown are predicted outcomes of oral infection of wild-type (C57BL/6J WT) (A) and B-cell-deficient (C57BL/6J B-cell KO) (B) mice with G. avium subsp. hominissuis (MAH) or M. avium subsp. paratuberculosis (MAP). Dots indicate bacillary burdens, and arrow widths are proportional to the numbers of translocated bacilli. In the model depicted, Grand. avium subsp. hominissuis is translocated mostly via enterocytes in WT mice.
What would be the evolutionary reward for M. avium subsp. paratuberculosis of crossing the intestinal mucosa by two different pathways? Nosotros hypothesize that the function of the 1000-prison cell pathway is to recognize and comprise the infection, while the enterocyte pathway represents the pathogenic road leading to dissemination. This hypothesis is consequent with the utilize of the enterocyte entry pathway by Grand. avium subsp. hominissuis, the intrinsically more virulent subspecies. The other plausible hypothesis is that each mechanism of entry represents a different outcome, both of which are important for the affliction. Interaction with the Peyer'southward patches would effect in rapid interaction with phagocytic cells, local spread, and an inflammatory response. In contrast, the infection of enterocytes would lead to a wearisome inflammatory response, translocation, and dissemination to distant sites, including the mammary glands. In the case of M. avium subsp. hominissuis, infection of enterocytes results in ho-hum translocation followed by faster systemic dissemination (21). Alternatively, cells of the dendritic cell lineage might play a function in the broadcasting of Chiliad. avium subsp. paratuberculosis. In fact, M. avium subsp. paratuberculosis infection of dendritic cells is inefficient. However, if the bacterium translocates across the epithelial barrier, uptake into dendritic cells increases 3- to vi-fold. This finding suggests that once bacteria cross the abdominal mucosa, they are readily ingested by dendritic cells. Once within dendritic cells, K. avium subsp. paratuberculosis was able to persist for 10 days, despite the lack of growth. Thus, dendritic cells may be responsible for the rapid spread of M. avium subsp. paratuberculosis to mesenteric lymph nodes, as observed by Wu and colleagues (39).
The finding that M. avium subsp. paratuberculosis translocates through enterocytes in our mouse model may also accept implications for the potential colonization of the human being epithelium. Recently, Golan et al. developed an M. avium subsp. paratuberculosis infection model using SCID mice transplanted with fetal human intestinal xenografts (7). Mice were infected past intraluminal challenge using a relatively depression dose (approximately 5 × ten7 CFU). The results indicated that goblet cells were predominantly infected compared to enterocytes. However, in that location are significant differences in the SCID model, including the use of homo xenografts, the inoculation route, the complete absence of both B and T cells, and a complex interplay of both mouse and man innate immune cells. These differences may explicate the predilection of M. avium subsp. paratuberculosis to invade either enterocytes or goblet cells. In this regard, intracellular pathogens, such every bit Salmonella spp., have been shown to invade both Yard cells and enterocytes, and, to a bottom extent, goblet cells, in mouse models, as reviewed past van Asten (34).
Our in vitro studies using polarized monolayers propose that binding and internalization of M. avium subsp. paratuberculosis do not cause a decrease in transmembrane resistance upwards to 24 h of infection. In add-on, the bacterium translocated through the epithelial monolayer, a finding that is in agreement with contempo observations by Wu and colleagues that M. avium subsp. paratuberculosis is found in the mesenteric lymph nodes of cows just 2 h later infection (39). Although surgery and intestinal clumping can be associated with increased permeability and can explain, in part, the results of Wu and colleagues, the observation appears to suggest that One thousand. avium subsp. paratuberculosis crosses the mucosal barrier. However, the percentage of bacteria that translocates is very small-scale. Histopathological observations of cattle infected with M. avium subsp. hominissuis and M. avium subsp. paratuberculosis indicate that while Chiliad. avium subsp. hominissuis spreads quickly from the intestinal mucosa, M. avium subsp. paratuberculosis disseminates very slowly (30), which, in fact, was observed in our system.
In conclusion, this study demonstrates that Chiliad. avium subsp. paratuberculosis uses both Peyer's patches and enterocytes to cross the intestinal mucosa, a machinery slightly different from that of 1000. avium subsp. hominissuis, which enters the mucosa preferentially by enterocytes (21). Yard. avium subsp. paratuberculosis also differs from K. avium subsp. hominissuis in the efficiency of dissemination. Further studies should focus on the understanding of those differences at the molecular level.
Acknowledgments
We give thanks Denny Weber for help with the preparation of the manuscript and Bernadette Stang for technical assistance.
We are also indebted to the Communicable diseases Foundation for the support of this research. L.E.B. was also supported by a grant from JDIP. R.K.B. was supported past funds from the BARD programme (IS-3673-05C), the Johne's Disease Integrated Plan (JDIP), USDA Cooperative State Service Project Pecker 14-141, and the School of Veterinary Medicine and Biomedical Sciences.
Footnotes
▿Published ahead of print on 24 May 2010.
REFERENCES
1. Alonso-Hearn, Chiliad., D. Patel, L. Danelishvili, Fifty. Meunier-Goddik, and L. Due east. Bermudez. 2008. The Mycobacterium avium subsp. paratuberculosis MAP3464 cistron encodes an oxidoreductase involved in invasion of bovine epithelial cells through the activation of host cell Cdc42. Infect. Immun. 76 :170-178. [PMC gratis commodity] [PubMed] [Google Scholar]
2. Bermudez, Fifty. East., M. Petrofsky, P. Kolonoski, and L. S. Immature. 1992. An animate being model of Mycobacterium avium circuitous disseminated infection afterwards colonization of the intestinal tract. J. Infect. Dis. 165 :75-79. [PubMed] [Google Scholar]
3. Bermudez, 50. E., F. J. Sangari, P. Kolonoski, M. Petrofsky, and J. Goodman. 2002. The efficiency of the translocation of Mycobacterium tuberculosis across a bilayer of epithelial and endothelial cells as a model of the alveolar wall is a consequence of transport within mononuclear phagocytes and invasion of alveolar epithelial cells. Infect. Immun. seventy :140-146. [PMC free article] [PubMed] [Google Scholar]
four. Bermudez, L. Due east., and Fifty. S. Young. 1994. Factors affecting invasion of HT-29 and HEp-2 epithelial cells past organisms of the Mycobacterium avium complex. Infect. Immun. 62 :2022-2026. [PMC free article] [PubMed] [Google Scholar]
5. Chacon, O., L. E. Bermudez, and R. G. Barletta. 2004. Johne'south disease, inflammatory bowel disease, and Mycobacterium paratuberculosis. Annu. Rev. Microbiol. 58 :329-363. [PubMed] [Google Scholar]
6. Damsker, B., and E. J. Bottone. 1985. Mycobacterium avium-Mycobacterium intracellulare from the abdominal tracts of patients with the acquired immunodeficiency syndrome: concepts regarding conquering and pathogenesis. J. Infect. Dis. 151 :179-181. [PubMed] [Google Scholar]
seven. Golan, 50., A. Livneh-Kol, Eastward. Gonen, South. Yagel, I. Rosenshine, and North. Y. Shpigel. 2009. Mycobacterium avium paratuberculosis invades human small-intestinal goblet cells and elicits inflammation. J. Infect. Dis. 199 :350-354. [PubMed] [Google Scholar]
8. Golovkina, T. V., M. Shlomchik, Fifty. Hannum, and A. Chervonsky. 1999. Organogenic office of B lymphocytes in mucosal immunity. Science 286 :1965-1968. [PubMed] [Google Scholar]
9. Hamilton, H. Fifty., D. K. Follett, L. Chiliad. Siegfried, and C. J. Czuprynski. 1989. Intestinal multiplication of Mycobacterium paratuberculosis in athymic nude gnotobiotic mice. Infect. Immun. 57 :225-230. [PMC free article] [PubMed] [Google Scholar]
10. Harris, Northward. B., and R. G. Barletta. 2001. Mycobacterium avium subsp. paratuberculosis in veterinary medicine. Clin. Microbiol. Rev. 14 :489-512. [PMC complimentary commodity] [PubMed] [Google Scholar]
11. Hines, One thousand. Due east., Two, J. R. Stabel, R. Due west. Sweeney, F. Griffin, A. M. Talaat, D. Bakker, M. Benedictus, West. C. Davis, G. W. de Lisle, I. A. Gardner, R. A. Juste, Five. Kapur, A. Koets, J. McNair, G. Pruitt, and R. H. Whitlock. 2007. Experimental challenge models for Johne'due south illness: a review and proposed international guidelines. Vet. Microbiol. 122 :197-222. [PubMed] [Google Scholar]
12. Inderlied, C. B., C. A. Kemper, and L. Eastward. Bermudez. 1993. The Mycobacterium avium circuitous. Clin. Microbiol. Rev. 6 :266-310. [PMC free article] [PubMed] [Google Scholar]
thirteen. Kim, S. Y., J. R. Goodman, M. Petrofsky, and Fifty. East. Bermudez. 1998. Mycobacterium avium infection of gut mucosa in mice associated with late inflammatory response and abdominal cell necrosis. J. Med. Microbiol. 47 :725-731. [PubMed] [Google Scholar]
fourteen. Mijs, W., P. de Haas, R. Rossau, T. Van der Laan, Fifty. Rigouts, F. Portaels, and D. van Soolingen. 2002. Molecular evidence to support a proposal to reserve the designation Mycobacterium avium subsp. avium for bird-type isolates and 'G. avium subsp. hominissuis' for the human being/porcine blazon of Chiliad. avium. Int. J. Syst. Evol. Microbiol. 52 :1505-1518. [PubMed] [Google Scholar]
fifteen. Momotani, Eastward., D. Fifty. Whipple, A. B. Thiermann, and N. F. Cheville. 1988. Role of 1000 cells and macrophages in the entrance of Mycobacterium paratuberculosis into domes of ileal Peyer'south patches in calves. Vet. Pathol. 25 :131-137. [PubMed] [Google Scholar]
sixteen. Mutwiri, One thousand. K., D. Chiliad. Butler, South. Rosendal, and J. Yager. 1992. Experimental infection of severe combined immunodeficient beige mice with Mycobacterium paratuberculosis of bovine origin. Infect. Immun. 60 :4074-4079. [PMC gratuitous article] [PubMed] [Google Scholar]
17. Patel, D., Fifty. Danelishvili, Y. Yamazaki, Grand. Alonso, Grand. L. Paustian, J. P. Bannantine, L. Meunier-Goddik, and Fifty. E. Bermudez. 2006. The power of Mycobacterium avium subsp. paratuberculosis to enter bovine epithelial cells is influenced by preexposure to a hyperosmolar environment and intracellular passage in bovine mammary epithelial cells. Infect. Immun. 74 :2849-2855. [PMC free commodity] [PubMed] [Google Scholar]
eighteen. Paustian, M. 50., V. Kapur, and J. P. Bannantine. 2005. Comparative genomic hybridizations reveal genetic regions within the Mycobacterium avium circuitous that are divergent from Mycobacterium avium subsp. paratuberculosis isolates. J. Bacteriol. 187 :2406-2415. [PMC gratis article] [PubMed] [Google Scholar]
nineteen. Petrofsky, M., and L. East. Bermudez. 2005. CD4+ T cells merely non CD8+ or γδ+ lymphocytes are required for host protection confronting Mycobacterium avium infection and dissemination through the intestinal road. Infect. Immun. 73 :2621-2627. [PMC free commodity] [PubMed] [Google Scholar]
20. Pott, J., T. Basler, C. U. Duerr, Grand. Rohde, R. Goethe, and 1000. W. Hornef. 2009. Internalization-dependent recognition of Mycobacterium avium ssp. paratuberculosis past abdominal epithelial cells. Cell. Microbiol. eleven :1802-1815. [PubMed] [Google Scholar]
21. Sangari, F. J., J. Goodman, Thou. Petrofsky, P. Kolonoski, and L. E. Bermudez. 2001. Mycobacterium avium invades the intestinal mucosa primarily by interacting with enterocytes. Infect. Immun. 69 :1515-1520. [PMC free article] [PubMed] [Google Scholar]
22. Sangari, F. J., K. Petrofsky, and L. E. Bermudez. 1999. Mycobacterium avium infection of epithelial cells results in inhibition or filibuster in the release of interleukin-eight and RANTES. Infect. Immun. 67 :5069-5075. [PMC complimentary article] [PubMed] [Google Scholar]
23. Secott, T. E., T. L. Lin, and C. C. Wu. 2004. Mycobacterium avium subsp. paratuberculosis fibronectin attachment poly peptide facilitates M-cell targeting and invasion through a fibronectin bridge with host integrins. Infect. Immun. 72 :3724-3732. [PMC gratuitous commodity] [PubMed] [Google Scholar]
24. Sigurdardóttir, O. G., A. Chiliad. Bakke-McKellep, B. Djonne, and O. Evensen. 2005. Mycobacterium avium subsp. paratuberculosis enters the small intestinal mucosa of goat kids in areas with and without Peyer'due south patches as demonstrated with the everted sleeve method. Comp. Immunol. Microbiol. Infect. Dis. 28 :223-230. [PubMed] [Google Scholar]
25. Sigurethardóttir, O. K., M. Valheim, and C. M. Printing. 2004. Establishment of Mycobacterium avium subsp. paratuberculosis infection in the intestine of ruminants. Adv. Drug Deliv. Rev. 56 :819-834. [PubMed] [Google Scholar]
26. Springer, B., L. Stockman, One thousand. Teschner, G. D. Roberts, and Eastward. C. Bottger. 1996. Ii-laboratory collaborative written report on identification of mycobacteria: molecular versus phenotypic methods. J. Clin. Microbiol. 34 :296-303. [PMC free article] [PubMed] [Google Scholar]
27. Streeter, R. N., Yard. F. Hoffsis, South. Bech-Nielsen, Westward. P. Shulaw, and D. Yard. Rings. 1995. Isolation of Mycobacterium paratuberculosis from colostrum and milk of subclinically infected cows. Am. J. Vet. Res. 56 :1322-1324. [PubMed] [Google Scholar]
28. Sweeney, R. Due west., R. H. Whitlock, and A. E. Rosenberger. 1992. Mycobacterium paratuberculosis cultured from milk and supramammary lymph nodes of infected asymptomatic cows. J. Clin. Microbiol. 30 :166-171. [PMC free article] [PubMed] [Google Scholar]
29. Taylor, T. One thousand., C. R. Wilks, and D. S. McQueen. 1981. Isolation of Mycobacterium paratuberculosis from the milk of a cow with Johne'southward disease. Vet. Rec. 109 :532-533. [PubMed] [Google Scholar]
xxx. Thoen, C. O., A. G. Karlson, and E. M. Himes. 1981. Mycobacterial infections in animals. Rev. Infect. Dis. three :960-972. [PubMed] [Google Scholar]
31. Thorel, M. F., Thou. Krichevsky, and V. V. Levy-Frebault. 1990. Numerical taxonomy of mycobactin-dependent mycobacteria, emended clarification of Mycobacterium avium, and description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. nov., and Mycobacterium avium subsp. silvaticum subsp. nov. Int. J. Syst. Bacteriol. 40 :254-260. [PubMed] [Google Scholar]
32. Tripathi, B. Northward. 2005. Paratuberculosis (Johne's disease) in cattle. Int. J. Cow Sci. i :16-26. [Google Scholar]
33. Turenne, C. Y., D. Yard. Collins, D. C. Alexander, and Thousand. A. Behr. 2008. Mycobacterium avium subsp. paratuberculosis and M. avium subsp. avium are independently evolved pathogenic clones of a much broader group of Thou. avium organisms. J. Bacteriol. 190 :2479-2487. [PMC free article] [PubMed] [Google Scholar]
34. van Asten, A. J., J. F. Koninkx, and J. Eastward. van Dijk. 2005. Salmonella entry: Chiliad cells versus absorptive enterocytes. Vet. Microbiol. 108 :149-152. [PubMed] [Google Scholar]
35. Wagner, D., F. J. Sangari, S. Kim, M. Petrofsky, and Fifty. E. Bermudez. 2002. Mycobacterium avium infection of macrophages results in progressive suppression of interleukin-12 production in vitro and in vivo. J. Leukoc. Biol. 71 :fourscore-88. [PubMed] [Google Scholar]
36. Weiss, D. J., C. D. Souza, O. A. Evanson, M. Sanders, and M. Rutherford. 2008. Bovine monocyte TLR2 receptors differentially regulate the intracellular fate of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium. J. Leukoc. Biol. 83 :48-55. [PubMed] [Google Scholar]
37. Whittington, R. J., and E. Southward. Sergeant. 2001. Progress towards agreement the spread, detection and control of Mycobacterium avium subsp. paratuberculosis in beast populations. Aust. Vet. J. 79 :267-278. [PubMed] [Google Scholar]
38. Wu, C. West., J. Glasner, M. Collins, S. Naser, and A. Grand. Talaat. 2006. Whole-genome plasticity among Mycobacterium avium subspecies: insights from comparative genomic hybridizations. J. Bacteriol. 188 :711-723. [PMC free article] [PubMed] [Google Scholar]
39. Wu, C. Westward., G. Livesey, South. Chiliad. Schmoller, E. J. Manning, H. Steinberg, Due west. C. Davis, Thousand. J. Hamilton, and A. M. Talaat. 2007. Invasion and persistence of Mycobacterium avium subsp. paratuberculosis during early stages of Johne'due south disease in calves. Infect. Immun. 75 :2110-2119. [PMC gratis article] [PubMed] [Google Scholar]
Manufactures from Infection and Immunity are provided hither courtesy of American Society for Microbiology (ASM)
What Do Peyer's Patches in Immunosuppressed Juvenile Animals Look Like
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2916292/
Comments
Post a Comment