Thomas J. Stabel

Immortalization and characterization of bovine peritoneal macrophages transfected with SV40 plasmid DNA

A transformed bovine peritoneal macrophage cell line was developed and characterized. Primary peritoneal macrophages were transformed by calcium-phosphate transfection with SV40 plasmid DNA. The transformed cell line retained the morphology of resident peritoneal macrophages as determined by light microscopy and histochemical analysis showed non-specific esterase activity. In addition, immunohistochemical staining of transformed peritoneal macrophages for lysozyme activity was positive. Transformed cells phagocytized Staphylococcus aureus, lysed chicken red blood cell (RBC) targets with and without opsonization and produced hydrogen peroxide radicals and interleukin-6 upon stimulation with opsonized zymosan and lipopolysaccharide, respectively. Transformed cells were also able to ingest and kill Mycobacterium paratuberculosis, an acid-fast bacillus. These results suggest that this cell line should be useful to study interactions between the bovine and intracellular pathogens.

Influence of inoculation route on the carrier state of Salmonella choleraesuis in swine

This study was designed to investigate the carrier state of swine infected with Salmonella choleraesuis. Thirty-five pigs were divided into 3 groups. Groups 1 (n = 15) and 2 (n = 16) were challenged with 10(8) CFU of S. choleraesuis intranasally or by gastric route, respectively. Group 3 (n = 4) served as uninoculated controls. Pigs were necropsied at 2, 4, 6, and 12 weeks post inoculation. Clinical signs and microscopic lesions were more severe for group 1. Salmonella choleraesuis was recovered from a greater percentage of tissue samples for group 1 versus group 2 at 2, 4, and 6 weeks post inoculation. No differences were observed between groups at 12 weeks post inoculation. Regardless of route of inoculation, S. choleraesuis was most often recovered from the ileocolic junction, ileocolic lymph node, cecal contents, tonsil, lung and colon. Both groups shed S. choleraesuis in the feces sporadically throughout the 12 week period indicating that a carrier state is maintained for at least 12 weeks. However, group 1 shed higher numbers of S. choleraesuis initially. Serum IgG, IgM, and IgA antibody responses to S. choleraesuis lipopolysaccharide and heat extract antigens were observed for both groups. Higher serum IgG antibody titers to S. choleraesuis lipopolysaccharide were observed for group 2. Intestinal antibody responses for both groups included IgG and IgM responses but not an IgA response. Both routes of inoculation stimulated peripheral blood B-cells while the intranasal route (group 1) was more effective at simulating peripheral blood T-cells. The reduction in levels of tissues infection and shedding observed for both groups coincided with the development of the host immune response. These data indicate that route of inoculation affects the development of humoral and cellular immunity, influences levels of Salmonella shed into the environment and the distribution of Salmonella within tissue.

Distribution of Anti-CD68 (EBM11) Immunoreactivity in Formalin-fixed, Paraffin-Embedded Bovine Tissues

A commercially acquired anti-human macrophage antibody (anti-CD68; EBM11) was used in an immunocytochemical technique to detect macrophages in formalin-fixed, paraffin-embedded tissues from cattle, pigs, humans, rats, turkeys, dogs, and cats. In healthy cattle, the antibody labeled alveolar macrophages, pulmonary intravascular cells (presumably intravascular macrophages), and macrophage-like cells in other tissues. In bovine lungs infected with Pasteurella haemolytica, EBM11 antibody labeled 95% of alveolar macrophages and macrophages within alveolar septa but only 0–2% of streaming or “oat” leukocytes. Alveolar macrophages were also stained by EBM11 in pigs but not in rats, turkeys, dogs, and cats. The antibody also stained macrophage aggregates in the mesenteric lymph nodes and intestinal lamina propria of Mycobacterium paratuberculosis-infected cattle. This study shows that the anti-CD68 (EBM11) antibody is a useful marker of macrophages in normal bovine tissues or tissues from areas of acute or chronic inflammation that have been routinely processed. The study also adds strength to the growing evidence suggesting that streaming leukocytes seen in pneumonic pasteurellosis are neutrophils.

Early Epithelial Invasion by Salmonella enterica Serovar Typhimurium DT104 in the Swine Ileum

Salmonella enterica serovar Typhimurium is an important intestinal pathogen in swine. This study was performed to document the early cellular invasion of Salmonella serovar Typhimurium in swine ileum. Ileal gut-loops were surgically prepared in ten 4- to 5-week-old mixed-breed pigs and inoculated for 0-60 minutes. Loops were harvested and prepared for both scanning and transmission electron microscopy (SEM and TEM, respectively). Preferential bacterial adherence to microfold cells (M cells) was seen within 5 minutes, and by 10 minutes bacterial invasion of the apical membrane was seen in M cells, goblet cells, and enterocytes. This multicellular invasion was observed throughout the course of infection. In addition, SEM revealed a specific affinity of Salmonella serovar Typhimurium to sites of cell extrusion. Using TEM, bacteria in these areas were focused in the crevices formed by the extruding cell and the adjacent cells and in the cytoplasm immediately beneath the extruding cell. Our results suggest that early cellular invasion by Salmonella serovar Typhimurium is nonspecific and rapid in swine. Furthermore, the combination of SEM and TEM data suggests that Salmonella serovar Typhimurium may use sites of cell extrusion as an additional mechanism for early invasion.

Segmented Filamentous Bacteria Interact with Intraepithelial Mononuclear Cells

ABSTRACT Segmented filamentous bacteria (SFB) are found in multiple species and play an important role in the development of mucosal immunity. The mechanism by which the bacteria interact with the immune system has not been well defined. We provide morphologic evidence of direct interaction between SFB and intraepithelial mononuclear cells.

Comparison of Early Ileal Invasion by Salmonella enterica Serovars Choleraesuis and Typhimurium

The mechanisms of Salmonella serovar-host specificity are not well defined. Pig ileal loops were used to compare phenotypic differences in early cellular invasion between non-host-adapted Salmonella serovar Typhimurium (SsT) and host-adapted Salmonella serovar Choleraesuis (SsC). By 10 minutes postinoculation, both serovars invaded a small number of M cells, enterocytes, and goblet cells. Multiple SsC organisms (up to 6 per cell) simultaneously invaded M cells, whereas SsT often invaded as one to two organisms per M cell. Internalization of both serovars resulted in vacuoles containing a single bacterium. The follicle-associated epithelium (FAE) of SsC-inoculated loops responded with more filopodia and lamellipodia although exhibiting less cell swelling than SsT. Additionally, SsT showed an enhanced affinity for sites of cell extrusion compared with SsC at 60 minutes. These results suggest: 1) both SsC and SsT exhibit non-cell-specific invasion as early as 10 minutes postinoculation, 2) Salmonella serovars exhibit differences in early invasion of FAE and M cells, and 3) cells undergoing extrusion may provide a site for preferential adherence by SsT and SsC.

Neutrophil Phagocytosis Following Inoculation of Salmonella choleraesuis into Swine

Neutrophils are an important mediator of host defence, especially in early stages of infection. A major function of neutrophils is the uptake and killing of invading microbes. Little is known about the effect of neutrophil activity on the pathogenesis and development of the carrier state in swine following infection with Salmonella choleraesuis. A human whole-blood microassay using flow cytometry was modified to measure the effect of S. choleraesuis infection in vivo on the rate of ingestion, or rate of uptake, of homologous bacteria by porcine neutrophils. Pigs were inoculated intranasally with 5–8×108 CFU S. choleraesuis and blood was collected in heparinized tubes at –5, 0, 1, 2, 3 and 4 days post inoculation (PI). Heat-killed S. choleraesuis were labelled with fluorescein isothiocyanate and incubated for various times with diluted whole blood. Red blood cells were lysed, external non-phagocytized bacteria were quenched with a commercially available lysing solution, and fluorescence from internalized bacteria labelled with fluorescein isothiocyanate was detected by flow cytometry. The rate of uptake by neutrophils did not increase until 2 days PI and then remained elevated to 4 days PI. The minimal uptake of S. choleraesuis early after exposure to these organisms may provide an opportunity for the pathogen to colonize and/or replicate to levels that facilitate establishment of a carrier state or clinical infection in swine.