堆型艾美耳球虫3-1E重组质粒微球的制备及体外释放试验

用聚乳酸羟基乙酸共聚物（PLGA）将鸡堆型艾美耳球虫重组表达质粒pcDNA3.1-3-1E包封,采用水包油包水（W/O/W）双重乳化方法制备pcDNA3-3-1E/PLGA微球。通过正交试验设计优化PLGA微球制备工艺,考察PLGA浓度、聚乙烯醇（PVA）浓度、超声功率、复乳搅拌时间对评价指标（即微球粒径大小、包封率、载药量）的影响,确定制备微球的最佳工艺条件。测定微球的形态、粒径、完整性、质粒DNA包封率、载药量和释放程度;进行体外模拟鸡胃液和肠液试验,观察微球体外释药效果。结果显示,当PLGA浓度为8%、PVA浓度为1.5%、超声功率为60W、复乳搅拌6h为微球制备的最佳工艺参数,在光镜下呈散在圆形,粒径小于12μm。微球的包封率、载药量分别为84.25%和4.46%,裸质粒与微球中质粒超螺旋比例差距不显著,这表明在包被过程中的超螺旋质粒未发生明显的降解。在模拟鸡的胃肠液累积释放试验中,它的累积释放能力依次为pH 3.0〉pH 7.4,载药微球在模拟鸡的胃肠液中24h释放26.8%,模拟肠液中24h释放11.2%,30d时体外累积释放率为81.7%,表明微球具有一定的缓释效果。     

Immune responses after local administration of IgY loaded-PLGA microspheres in gut-associated lymphoid tissue in pigs

Oral vaccination of large animals using PLGA MS (poly(D,L-lactide-co-glycolide)microspheres) appeared to be more challenging than immunization of mice. The purpose of this study was to deliver to GALT an immunogenic model protein (IgY), free or encapsulated by spray-drying in PLGA MS, and to evaluate systemic immune response in SPF Large White pigs. Pigs were surgically processed for local administration of IgY in three sets of experiments. In two sets of experiments, administration was locally performed in temporary ligatured intestinal segments, in jejunal Peyer's patches and in mesenteric lymph nodes. In the third experiment, pigs received IgY via an intestinal cannula. Total IgY-specific antibodies were detected in the sera of pigs after a single local immunization, but not in the sera of cannulated pigs. The study of IgG1 and IgG2 isotypes indicated that PLGA MS are able to elicit a combined serum IgG2/G1 response with a predominance of IgG1 response when locally administered. PLGA MS can be a potential oral delivery system for antigen but our results underlined the difficulty to immunize large animals like pigs. Transposition of data between small and large animals appears to be complex and suggests that physiological features need to be considered to increase intestinal availability of oral encapsulated vaccines.     

Induction of mucosal immune responses following enteric immunization with antigen delivered in alginate microspheres

Oral immunization is the most effective way of inducing immune responses in the intestinal tract. Biodegradable microspheres have been used extensively for the delivery of antigens to the Peyer's patches (PPs) within the gut-associated lymphoid tissue (GALT). We evaluated various formulations of alginate microspheres for their capacity to induce mucosal immune responses in vivo. Multiple intestinal "loops" each containing a single PP, were surgically prepared in lambs. We have previously showed that PP in individual intestinal loops function as independent sites for the induction of immune responses. This animal model provides a system for directly comparing different antigen formulations within the same animal. Individual intestinal loops were injected with a model antigen, porcine serum albumin (PSA) encapsulated in three different formulations of alginate micropsheres. Three weeks after immunization, PSA-specific immune responses were assayed with antibody secreting cell (ASC) ELISPOT, lymphocyte proliferative responses (LPRs), IFN-gamma production and antibody secreted into intestinal loops. PSA encapsulated in alginate micropsheres or in saline induced humoral immune responses as indicated by the presence of numerous ASC. However, PSA-specific T-cell responses (LPR and IFN-gamma production) were not induced.     

Production and characterization of biodegradable Povidone-iodine microsphere as a intramammary disinfectant

Microspheres composed of biocompatible, biodegradable poly DL-lactide-co-glycolide (DL-PLGA) and Povidone-iodine were evaluated as an intramammary disinfectant delivery system in vitro prior to infusion into mammary glands. Microsphere was prepared by solvent evaporation method and particle size, morphology and in vitro release kinetics were examined. The microspheres were ranged in size from 25 microm to 155 microm (mean diameter = 65.7 microm). Povidone-iodine was dispersed on the surface of microsphere and microsphere was spherical in shape with a smooth surface. The yield of microsphere was 57.3% and the encapsulation efficiency was 69.6%. In in vitro release study, a burst effect (50.9%) was observed during the first two days and a sustained release then continued for the next 28 days. Results of the present study demonstrated that microsphere have the potential for new intramammary disinfectant formulations that can provide increased efficacy of therapy against mastitis pathogens.     

Mycobacterium avium subsp. paratuberculosis enters the small intestinal mucosa of goat kids in areas with and without Peyer's patches as demonstrated with the everted sleeve method

The main lesions of paratuberculosis in ruminants are in the small intestine. Previous studies have shown that the bacterium enters the small intestine through M cells found in the follicle-associated epithelium lining the domes of the Peyer's patches. The everted sleeve method, devised for the in vitro study of intestinal absorption, was used in this study to investigate the uptake of Mycobacterium avium subsp. paratuberculosis in goat intestine. Everted small intestinal sleeves of goat kids, prepared from areas with and without Peyer's patches, were incubated for 60 min in 3H-labeled bacterial solution. The results of this study imply that the bacteria can enter the intestinal mucosa of the jejunum, both in areas with and without Peyer's patches. These findings indicate, therefore, that M. avium subsp. paratuberculosis bacteria not only enter through M cells but also through enterocytes.     

Immunobiotic Lactobacillus strains augment NLRP3 expression in newborn and adult porcine gut-associated lymphoid tissues

We isolated cDNA encoding porcine nucleotide-binding domain-like receptor family, pryin domain containing 3 (NLRP3) from Peyer's patches. The complete nucleotide open reading frame of porcine NLRP3 contains 3108-bp encoding a deduced polypeptide of 1036-amino acid residues. The porcine NLRP3 amino acid sequence is more similar to the longest isoform of human than the mouse counterpart. The predicted amino acid sequence of porcine NLRP3 presented nine C-terminal leucine-rich repeat domains. In newborn swine, the expression of NLRP3 was detected at higher levels in spleen and mesenteric lymph nodes, while lower levels were observed in intestinal tissues. In adult swine, NLRP3 was strongly expressed in Peyer's patches and the mesenteric lymph nodes, and the expression level in the lower intestinal tissues was comparable to that in spleen. Toll-like receptor and nucleotide-binding domain ligands, as well as Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus gasseri, enhanced NLRP3 expression in gut-associated lymphoid tissues (GALT) of newborn and adult swine. Our results should aid in understanding the intestinal immunoregulatory mechanisms underlying NLRP3 activation and the priming ability of immunobiotic lactic acid bacteria in porcine GALT.     

Morphology of intestinal colonization of Yersinia enterocolitica serovar O3 in mice

The present study was made to know the morphology of the initial invasion and lesions involved in the intestinal colonization of Yersinia enterocolitica serovar O3 in the epithelium of Peyer's patches of mice. Microfold (M) cells formed a specific structure like a pseudopodium and the bacteria were observed on the surface of the pseudopodium-like structure 4 hr after oral administration of serovar O3. The colonies of serovar O3 were observed in the epithelium and the lamina propria of the Peyer's patches dome region, and the bacteria grown in the Peyer's patches were in direct contact with the lumen without covered with the host tissue 24 hr after the administration.     

Uptake of colostral leukocytes in the intestinal tract of newborn calves

The role of colostral immunoglobulins for the protection of newborn calves has been studied extensively, but little is known about the importance of colostral leukocytes. To study the uptake of colostral leukocytes in the intestine of calves and to determine preferential sites for this uptake, FITC-labelled colostral cells derived from the respective dams were injected into intestinal loops with/without Peyer's patches of three male Holstein Frisian calves about 5h post natum. In adjacent loops, PBS was injected as control. Loops were excised after an exposure of 1.5-2h. FITC-labelled material and cells were detected by the direct immunoperoxidase method in paraplast sections. Twenty-five consecutive sections were evaluated from each localization. Uptake of labelled material and cells was observed in all three calves in the jejunal Peyer's patch and in two calves in the ileal Peyer's patch as well. In the jejunal Peyer's patch, labelled material and cells were present in epithelium, domes and sinuses around lymphoid follicles, whereas in the ileal Peyer's patch, they were found in the sinuses only. These findings confirm that uptake of colostral leukocytes through the intestinal barrier is possible and that the preferential route of uptake is through follicle-associated epithelium of Peyer's patches.     

Tissue distribution of bovine spongiform encephalopathy infectivity in Romney sheep up to the onset of clinical disease after oral challenge

Sixty Romney sheep of three prion protein genotypes were dosed orally at six months of age with an inoculum prepared from the brains of cattle clinically affected with BSE, and 15 sheep were left undosed as controls. They were randomly assigned within genotype to groups and were sequentially euthanased and examined postmortem at intervals of six or 12 months, depending on their predicted susceptibility. Tissue pools prepared from the three, four or five dosed animals in each group were inoculated into groups of 20 RIII mice as a bioassay for infectivity. Separate inocula were prepared from the matched control sheep killed at each time. In the ARQ/ARQ sheep killed four months after inoculation, infectivity was detected in the Peyer's patch tissue pool, and at 10 months it was detected in the spleen pool; from 16 months, infectivity was detected in a range of nervous and lymphoreticular tissues, including the spinal cord pool, distal ileum excluding Peyer's patches, liver, Peyer's patches, mesenteric and prescapular lymph nodes, spleen, tonsil and cervical thymus. No infectivity was detected in the tissue pools from the ARQ/ARR and ARR/ARR sheep killed 10 months or 22 months after infection.     

Lymphocyte migration in the intestinal mucosa: entry, transit and emigration of lymphoid cells and the influence of antigen

Lymphocyte migration is important to transport immunological information between the different compartments of the intestinal immune system. Large numbers of lymphocytes emigrate from the Peyer's patches and reach the blood circulation after expansion and maturation within the mesenteric lymph nodes. So far the frequency of antigen specific lymphocytes emigrating from the Peyer's patches after oral stimulation is not known. After mesenteric lymph node resection those cells emigrating from the intestinal wall are accessible by calculating the major intestinal lymph duct. The first antigen specific cells draining from the intestines are obviously not lymphocytes but dendritic cells, thus the antigen is rapidly trapped in the parenchyma of the lymph nodes in vivo. When lymphocytes were taken from intestinal lymph, labeled in vitro and retransfused, marked numbers of B-cells were re-detected in intestinal lymph. Later preferentially T-cells recirculated through the gut wall. After immigration into the intestinal lamina propria the lymphocytes may enter the space between epithelial cells, where they are present as intraepithelial lymphocytes. Lymphoid cells in the S-phase of the cell cycle have been detected in all compartments of the intestinal wall. Apoptosis is probably a further important mechanism for the regulation of intestinal immunity in removing cells reacting against harmless dietary antigens to maintain oral tolerance.     

Uptake of Mycobacterium avium subsp. paratuberculosis through the distal small intestinal mucosa in goats: an ultrastructural study

Various pathogens gain access to the intestinal wall via specialized cells, the M cells, found among the follicle-associated epithelial cells overlying the domes of the Peyer's patches. The present study was undertaken to examine the uptake of live Mycobacterium avium subsp. paratuberculosis in the distal small intestine of goat kids. Following laparotomy, distal small intestinal segments of five goats were ligated and injected with bacterial suspension. After 1 hour, the intestinal segments were excised and fixed for light and electron microscopic studies. M. a. paratuberculosis organisms were observed by transmission electron microscopy at locations in the intestinal wall, suggesting transcellular transportation through the M cells. The organisms were present both in the cytoplasm of the M cells and in the cytoplasm of intraepithelial leukocytes found in M-cell pockets. Intercellular bacteria between M cells were occasionally seen. Bacteria were not observed in association with the absorptive epithelium. This study indicates that in goat kids, M. a. paratuberculosis enters the intestinal wall primarily through the M cells in the follicle-associated epithelium of the Peyer's patches.     

The intestinal habitat for organized lymphoid tissues in ruminants; comparative aspects of structure, function and development

Unlike the Peyer's patches of rats and mice, which are considered to be secondary lymphoid organs, the ileal Peyer's patch of sheep is thought to be responsible for the primary generation of B cells, like the bursa of Fabricius of birds. The ileal Peyer's patch of sheep shows prenatal maturation, antigen-independent lymphopoiesis, a rate of lymphocyte production larger than that of the thymus, and involution at a young age. Follicles contain few T cells and have an IgM+, relatively immature B lymphocyte population, as judged by B-cell differentiation markers. The follicle-associated epithelium of the ileal Peyer's patch is of a special type that sheds carbonic anhydrase-rich, 50-nanometer membrane-bounded particles (carbonic anhydrase-reactive particles; CAP) into the intercellular spaces. The CAP filter into the follicle centre and are taken up by lymphocytes. They represent the epithelial (bursa-like) element in an otherwise mesenchymal stroma of reticular cells embedding the follicle lymphocytes. Transepithelial transport of macromolecules, with the formation of multivesicular body-like cytoplasmic vacuoles, appears to be the basis for CAP formation. The jejunal Peyer's patches are devoid of CAP, persist in the adult animal, contain M cells with clusters of B cells in the follicle-associated epithelium, and have many CD4+ lymphocytes in the follicles and in the interfollicular areas. Aggregates of lymphoid follicles in the large intestine resemble the jejunal Peyer's patches with respect to their lymphocyte population and the ileal Peyer's patch with respect to their follicle-associated epithelium.     

Characteristics of lactoferrin receptor in bovine intestine: higher binding activity to the epithelium overlying Peyer's patches

Several lines of evidence have recently demonstrated the occurrence of specific lactoferrin (Lf) receptors in different cells. We report here, for the first time, the characteristics of binding, and distribution of Lf receptors in the bovine intestinal tract with special emphasis on the epithelium overlying Peyer's patches (EOPP). Brush-border membrane vesicles (BBMV) were prepared from the mucosa of duodenum, jejunum, ileum, colon, EOPP in jejunum and EOPP in ileum. Receptor binding assays were carried out using 125I-labelled bovine Lf. Specific and saturable Lf receptors were found in BBMV of all the intestinal segments examined. Non-linear regression and Scatchard plot analyses clearly revealed that EOPP had the highest binding maximal (Bmax), and lowest in colon. The maximum dissociation constant (Kd) 3.74 microm was in the ileum. We found that bovine transferrin competed with Lf for the same binding site of receptors. In contrast, no binding of bovine serum albumin occurred. It was concluded that Lf receptors in the mucosal lining are attributable to mediate multifunctional activities of Lf in the gut, especially in the EOPP.     

Age-related changes in the anatomical characteristics of Peyer's patches in small intestine of Bactrian camels (Camelus bactrianus)

The distribution, size, and appearance of Peyer's patches vary according to species. In order to determine the anatomical characteristics of Peyer's patches in small intestine of Bactrian camel, and age-related changes in the number of Peyer's patches, 40 Bactrian camels of the following four age groups were studied: young (0.5–2 years), pubertal (3–5 years), middle-aged (6–16 years), and old (17–20 years). The exact number of Peyer's patches was recorded, and the appearance of Peyer's patches was described in detail. The results indicated that Peyer's patches of Bactrian camels not only have a particular anatomical location and distinct appearance but also change with age. They were distributed in the whole small intestine and there were four distinct types of Peyer's patches: nodular, faviform, cup-shaped, and cystic form Peyer's patches. However, the nodular and cystic form Peyer’s patches are specific to Bactrian camel, which have not been found in other animals including Dromedary camel. In addition, the distribution density of Peyer's patches in ileum was the maximum, then was jejunum and duodenum. Further statistical analysis showed that the number of Peyer's patches was altered with age. The number peaked in 5-year-old camels and declined subsequently with age. However, there was little change in the size of Peyer's patches in different age groups; no age-related macroscopic variations in the shape or size of the Peyer's patches were found. Results obtained from this study provide the basic information to further study on the gastrointestinal mucosal immunity of Bactrian camel.     

Effects of spray-dried porcine plasma and plant extracts on intestinal morphology and on leukocyte cell subsets of weaned pigs

We evaluated the effects of a 6% spray-dried porcine plasma (SDPP) and a plant extracts mixture (XT; 5% carvacrol, 3% cinnamaldehyde, and 2% capsicum oleoresin) on the productive performance, intestinal morphology, and leukocyte cell subsets of early-weaned pigs compared with a control group. Morphometry of the jejunum, ileum, and colon, and immune cell analysis of blood, ileocolic lymph node (LN), and ileal Peyer's patches were done in 24 weaned pigs (20 +/- 2 d) at 19 or 21 d postweaning. Although SDPP and XT treatments did not increase ADG or ADFI, SDPP improved the G:F ratio (P = 0.024) compared with the control group. Dietary SDPP reduced the percentages of blood monocytes (P = 0.006) and macrophages in ileal Peyer's patches and LN (P = 0.04), of B lymphocytes (P = 0.04) and gammadelta+ T cells in LN (P = 0.009), and of intraepithelial lymphocytes (P = 0.026) as well as the density of lamina propria cells in the colon (P < 0.01). Dietary XT reduced intraepithelial lymphocyte numbers in jejunum (P = 0.034) and the percentages of blood cytotoxic cells (P = 0.07) and B lymphocytes in LN (P = 0.03); however, XT increased blood monocytes (P = 0.038) and the density of lamina propria lymphocytes in the colon (P = 0.003). These results indicate that dietary SDPP and plant extracts can affect intestinal morphology and immune cell subsets of gut tissues and blood in weaned pigs. Furthermore, the effects of SDPP suggest lower activation of the immune system of the piglets.     

Sequential development of histologic lesions and their relationship with bacterial isolation, fecal shedding, and immune responses during progressive stages of experimental infection of lambs with Mycobacterium avium subsp. paratuberculosis

Understanding pathogenesis during progressive stages of infection by Mycobacterium avium subsp. paratuberculosis (MAP) and finding suitable methods for its diagnosis are key to the control of Johne's disease in animals. Paratuberculosis was experimentally produced in 20 crossbred lambs by oral administration of MAP to study the sequential development of lesions between 10 and 330 days postinfection and to assess commonly used diagnostic methods such as bacterial culture, lymphocyte stimulation test (LST), and enzyme-linked immunosorbent assay (ELISA) during progressive stages of infection. Histologic lesions were classified into four grades from grade 1 (least severe) to grade 4 (most severe) on the basis of location of granulomatous lesions in different regions and layers of intestines, their association with intestinal lymphoid tissues, pattern and distribution of lesions, types of cellular infiltration, and presence of acid-fast bacilli. It is evident that infection first establishes in lymphoid tissues of the small intestine, possibly at multiple sites, producing segmental lesions and from there spreads to lamina propria and local lymph nodes. Wide variability in the histologic lesions in relation to postinfection periods and initial tropism of MAP to the intestinal lymphoid tissues (Peyer's patches) suggests a differential susceptibility of young animals, possibly because of compositional phenotypic variation of Peyer's patches influencing subsequent course of infection. Histopathology was found to be a better indicator of paratuberculous infection than bacteriology in sheep. The LST (reflecting the cellular immune response) and ELISA (reflecting the humoral immune response) had overall sensitivities of 65% (11 of 17) and 42% (8 of 19), respectively, in sheep with different types of pathology but when employed together could detect about 88% of infected animals.     

Follicle associated epithelium of the gut associated lymphoid tissue of cattle

The morphology of the gut-associated lymphoid tissue of the small and large intestine in three gnotobiotic calves was examined by scanning and transmission electron microscopy, and the distribution of specialized membranous cells present in the follicle associated epithelium was defined. Isolated follicles remaining in the ileum of a cow after involution of the continuous Peyer's patch were examined by scanning electron microscopy. The presence of membrane-bound particles, reported to be exclusively associated with the continuous Peyer's patch, was investigated in other gut-associated tissue of the small and large intestine of the calf. The presence of two types of follicle associated epithelium in the small intestine of the calf was confirmed, and the follicle associated epithelium of the large intestine proved to be a homogeneous population of specialized membranous cells, similar to that of the continuous Peyer's patch of the small intestine. In the discrete Peyer's patches, some specialized membranous cells were completely hidden by adjacent enterocytes and could only be identified by cytoplasmic extensions into the intestinal lumen. In the proximal part of the continuous Peyer's patch, a transitional zone was detected where the follicle associated epithelium of some doomed villi was composed of a homogeneous population of specialized membranous cells, while the epithelium covering other doomed villi consisted of a mixture of absorptive and specialized membranous cells, usually only found in the discrete Peyer's patches. Membrane-bound particles were observed associated with gut-associated lymphoid tissue in the small and large intestine.     

Gene discovery and expression profiling in porcine Peyer's patch

Peyer's patches of the intestinal mucosa are essential for host defense and immune regulation in the enteric system. To better understand molecular mechanisms of Peyer's patch function, we have screened for differentially expressed genes specific to Peyer's patch. cDNA libraries were created from normal Peyer's patch, immune stimulated Peyer's patch, and pooled cDNA subtracted with fibroblast RNA. From the subtracted library, 3687 expressed sequence tags (ESTs), representing 2414 unique nucleotide sequences, were isolated, identified by BLAST searches against public databases, and spotted onto a microarray for gene expression profiling. Approximately 30% of these ESTs BLAST to genes of unknown function and 20% have no known homology in the public databases (novel genes). Of the novel genes, 70% are expressed in normal immune tissues by microarray analysis, suggesting that at least 371 of the unidentified EST sequences from the subtracted library are novel porcine genes and can now be further characterized to determine their function in the porcine Peyer's patch. We surmise that the products of these genes participate in biochemical and cellular functions related to the unique immunological and gastroenterological functions of the small intestine. The BLAST and gene ontology information for each of the subtracted library EST sequences, the normal and immune stimulated libraries, and the microarray are all valuable resources that will facilitate further examination of the biological function of porcine Peyer's patch tissue.     

Characterization and Distribution of B Cells in the Lymphoid Organs of Goats

The distribution of B cells in the lymphoid organs of the goat was studied using a panel of 13 monoclonal antibodies (mAbs) developed against different markers for bovine B cells. Samples of mesenteric lymph nodes, jejunal and ileal Peyer's patches, duodenum, jejunum, ileum, colon, caecum and rectum were taken from four 7-month-old male Murciano-granadina goats using the avidin-biotin-peroxidase (ABC) method on frozen sections as described by Hsu et al. (1981). The mAbs against immunoglobulins (Ig) recognized a large number of cells, particularly in the light zones of the germinative centres of the lymphoid follicles, regardless of the isotype against which they were directed. However, the greatest numbers of B cells in the germinative centres and outer coronas of the lymphoid follicles of the lymph node, spleen and Peyer's patches were recognized by mAbs against the L lambda chain of Ig and against IgM. This was also the case in other locations where B cells were abundant, such as the medulla of the lymph node and the dome of the Peyer's patches. These mAbs recognized not only B lymphocytes but also plasma cells, showing an intracytoplasmatic reaction (numerous in the spleen red pulp and the intestinal lamina propria when mAbs were used against the L lambda chain of the Ig, scarce in the intestinal lamina propria when used against IgM and scarce in spleen red pulp and numerous in the intestinal lamina propria when mAbs against IgA were used). The mAbs BAQ44 A, GC65 A and GB25 A are of interest because, besides marking cells in the B areas where lymphocytes show surface Ig, they give a positive reaction in areas where there are Ig-cells (the dark zone of the germinative centre) and do not immunostain plasma cells. Thus, these mAbs recognize a surface marker which is not an Ig.     

Direct inoculation of Mycobacterium avium subspecies Paratuberculosis into ileocecal Peyer's patches results in colonization of the intestine in a calf model

The objective of this study was to develop an intestinal model of Mycobacterium avium subspecies paratuberculosis (Map) infection in the calf for evaluation of mucosal pathology and local and systemic immunologic responses. Map was inoculated into Peyer's patches of young calves using a right flank surgical approach in standing calves to exteriorize the ileocecal junction. Inoculum doses ranging from 10(3) to 10(9) colony-forming units of strain K10 Map were injected through the serosal surface into Peyer's patches of the distal ileum near the ileocecal valve. Fecal samples were collected for culture from each calf weekly until termination of the study. Calves were necropsied at 7, 30, 60, and 90 days after infection, when inoculation sites, lymph nodes, spleen, and peripheral blood were collected for evaluation. Ileocecal lymph nodes were consistently colonized by Map in the 10(5) to 10(9) groups. The ileocecal valve was also colonized in 10(7) and 10(9) groups. This correlated with fecal culture results as infected calves intermittently shed Map in their feces throughout the study. Granulomatous lesions with giant cells and acid-fast bacilli at the ileocecal junction, ileocecal lymph nodes, and lamina propria of high-dose animals (10(7) and 10(9)) were identified from each time point. Flow cytometry was used to detect antigen-specific production of interferon-γ and interleukin-4 locally (ileocecal lymph node) and systemically (peripheral blood mononuclear cells), which defined distinct immunologic profiles in low-dose and high-dose calves. This study demonstrates intestinal Map infection via Peyer's patch inoculation, a novel model with many shared features of natural Map infection.