Use of porcine tumor necrosis factor receptor 1-Ig fusion protein to prolong xenograft survival.

BACKGROUND: Delayed rejection of xenografts is a major hurdle that needs to be addressed to achieve long-term engraftment in the pig-to-primate transplant setting. Both vascular and avascular xenografts are susceptible to a delayed rejection process that comprises humoral and cellular responses. Tumor necrosis factor (TNF) is believed to play a role in this process by promoting cell activation, apoptosis and the recruitment of inflammatory cells. To address this problem, we engineered the donor cell in such a way that it could block both human and porcine TNF. METHODS: We produced a recombinant fusion protein containing the extracellular domain of the porcine TNF-Receptor 1 and an IgG Fc moiety (pTNFR1Ig). We first evaluated by flow cytometry the pTNFR1Ig capacity to prevent TNF alpha-induced expression of SLAI, SLAII, VCAM-1, ICAM-1 and E-selectin on the cell surface of porcine aortic endothelial cells (PAEC). The effect on TNF alpha-mediated cell death was also assessed by propidium iodide staining after incubating PAEC with TNF alpha plus cycloheximide for 24 h. PAEC and porcine fibroblasts were subsequently engineered by retroviral infection to express and secrete pTNFR1Ig and their resistance to the TNF alpha effects was tested in vitro. Finally, we transplanted mock-control and pTNFR1Ig-expressing PAEC under the kidney capsule of BALB/c mice in the absence of immunosuppression and examined the degree of rejection at 2 and 3 weeks post-transplantation. RESULTS: Treatment with pTNFR1Ig resulted in a very potent blockade of human, porcine and murine TNF alpha activity on porcine cells. It inhibited the upregulation of all cell surface markers of activation tested as well as the TNF alpha-mediated cell death. Moreover, pTNFR1Ig-expressing PAEC showed prolonged engraftment in a pig-to-mouse xenotransplant model. CONCLUSIONS: Incorporation of strategies that block TNF may prove useful in the development of xenografts resistant to delayed rejection.

Effect of 2-deoxy-d-glucose induced stress on Salmonella choleraesuis shedding and persistence in swine.

A glucose analog, 2-deoxy-d-glucose (2DG), previously shown in swine to induce many of the hallmark parameters of stress, was administered to Salmonella choleraesuis carrier-swine and the effects on Salmonella fecal shedding and tissue colonization were evaluated. Initially, pigs were divided into two groups, one that received 1 x 10 (6) S. choleraesuis and one group that received saline. At 3 or 6 weeks post inoculation (PI), half of each group received an injection of 2DG and the other half received saline. Throughout the study, individual fecal samples were collected and quantitatively cultured for Salmonella, tonsil and nasal swabs were qualitatively cultured, clinical signs were monitored, temperatures were measured and whole blood collected. Pigs were necropsied 8-18 days after 2DG treatment. The experimental stress induced by 2DG was not sufficient to cause recrudescence of Salmonella fecal shedding even when tissues were culture positive for Salmonella. In addition, persistent shedding was not affected by 2DG administration. Although the complex set of parameters that constitute the stress phenomenon is still relatively unknown, it is now apparent that the stressful event(s) sufficient to trigger Salmonella recrudescence involves more than just increased blood glucose, increased cortisol, and inhibition of lymphocyte proliferation.

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.

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.

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 x 10(8) 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.

Secretion of a putative cytotoxin in multiple antibiotic resistant Salmonella enterica serotype Typhimurium phagetype DT104.

Salmonella enterica serotype Typhimurium phagetype DT104 (DT104) is a multiple antibiotic-resistant pathogen. DT104 infections have been reported in a multitude of hosts including humans, companion animals, livestock and wildlife. Recently, several isolates of DT104 were recovered from veal calves exhibiting abomasitis, a finding that is inconsistent with classic salmonellosis. One of these isolates was used in murine ligated loop experiments where it was observed that multiresistant DT104 can elaborate a putative cytotoxin. Thus it appears that DT104 has the ability to evade pharmacologic interventions, via antibiotic resistance, and elaborate a toxin that can damage cells.

The effect of dietary soy genistein on pig growth and viral replication during a viral challenge.

Twelve replications of four littermate pigs from a herd naive for porcine reproductive and respiratory syndrome (PRRS) were weaned (10 +/- 2 d of age) and penned individually in isolation rooms. Pigs were randomly allotted within litter to one of four dietary soy genistein concentrations (0, 200, 400, and 800 ppm) to quantify the effect of soy genistein on pig growth and virus replication during a viral challenge. Genistein was provided as the soy glycoside, genistin. At 29 +/- 2 d of age (4.9 +/- 1.4 kg BW), pigs were oronasally inoculated with 10(4.3) PRRS virus/mL from strain JA142 in a 2-mL dose. Blood was collected every 4 d from d 0 to 24 postinoculation and analyzed for serum PPRS virus, interferon activity, and alpha1-acylglycoprotein (AGP) concentrations. Serum virus and interferon peaked at 10(5) virus/mL and 57% protection, respectively, at 4 d postinoculation and then declined steadily. Serum AGP concentration peaked at 12 d postinoculation. Each log increase in serum virus was associated with a reduction of daily gain of 0.034 kg in 5.3-kg pigs and 0.004 kg in 11-kg pigs. As dietary genistein concentration increased, serum concentrations of PRRS virus decreased linearly (10(2.46), 10(2.26), 10(2.05), and 10(2.14) virus per milliliter of serum, P < 0.07) and interferon responded quadratically (28.4, 25.7, 22.8, and 30.9% protection, P < 0.06) independent of days postinoculation. The AGP concentrations increased (P < 0.01) quadratically with the magnitude of the response to dietary genistein maximized at 12 to 16 d postinoculation. Effects of dietary genistein on daily pig gain and feed intake were dependent on dietary genistein concentration and stage of viremia. Daily pig gains from d 0 to 24 postinoculation were improved as dietary genistein increased, but the magnitude of the response to dietary genistein concentration lessened as the serum virus concentrations were minimized resulting in a linear genistein x period interaction (P < 0.07). Daily feed intakes also were increased quadratically as genistein concentration increased. These data indicate that soy genistein at dietary concentrations of 200 to 400 ppm is an orally active immune modulator that enhances systemic serum virus elimination and body growth in virally challenged pigs.

The effect of dietary soy daidzein on pig growth and viral replication during a viral challenge.

Twelve replications of four littermate pigs from a porcine reproductive and respiratory syndrome (PRRS) naive herd were weaned (11 +/- 2 d of age) and penned individually in isolation rooms. Pigs were randomly allotted within litter to one of four dietary soy daidzein concentrations (0, 200, 400, or 800 ppm) to quantify the effect of daidzein on growth and immune response during a PRRS challenge. Daidzein was provided as the soy aglycone. At 27 +/- 2 d of age (4.9 +/- 1.4 kg BW), pigs were oronasally inoculated with 10(4.3) PRRS virus/mL from strain JA142 in a 2-mL dose. Blood was collected every 4 d from d 0 to 24 after inoculation and analyzed for serum PRRS virus, interferon, and alpha-1-acylglycoprotein (AGP) concentrations. Serum virus and interferon peaked at 10(5.3) virus/mL and 79% protection, respectively, at 4 d after inoculation and then declined steadily. Serum AGP concentration peaked at 12 d after inoculation. Each log increase in serum virus was associated with an increase in serum interferon, which resulted in a decrease of pig ADG and daily feed intake of 0.019 kg and 0.023 kg, respectively, in 5.8-kg pigs and a feed intake reduction of 0.024 kg in 12.5-kg pigs. Dietary daidzein additions did not (P > 0.3) alter the serum concentration after inoculation of PRRS virus (10(1.79), 10(1.94), 10(1.86), 10(1.93) virus/mL of serum) or AGP. Serum concentrations of interferon responded cubically (30.3, 28.9, 29.4, and 31.1% protection) as dietary daidzein concentrations increased; however, the magnitude of the response decreased over time. Dietary daidzein additions resulted in improvements in daily pig gain, daily feed intake, and gain/feed during periods of peak viremia (d 4 to 16 after inoculation), but not in periods when systemic virus concentrations were minimized (d 16 to 24 after inoculation), resulting in a daidzein x days after inoculation interaction. Based on these data, the magnitude of the growth responses that occur in pigs infected with a virus is quantitatively related to the animal's serum concentration of the virus and interferon, and dietary soy daidzein at 200 or 400 ppm is a weak enhancer of body growth in virally challenged pigs.

A simple and rapid flow cytometric method for detection of porcine cell surface markers.

The objective of this study was to develop a rapid and reliable method for flow cytometric analysis of porcine whole blood cells. Fifty-microliters of heparin- or EDTA-treated whole blood was added to wells of a round-bottom 96-well microtitration plate. Each well contained 10 microl of an appropriate dilution of four different antibodies (40 microl total; two primary monoclonal antibodies and two fluorescent-labeled secondary antibodies). For convenience, the antibody mixture could be added to plates 1-2 days prior to assay and stored at 4 degrees C. Once whole blood was added to wells, plates were mixed gently, placed in a sealed bag and incubated in the dark at room temperature for 20 min. Contents of wells were then transferred to polystyrene tubes containing 2 ml of 1.5% formalin in distilled water and mixed gently. Cells were fixed for a minimum of 30 min and then stored in the dark at 4 degrees C until analysis by flow cytometry. Analysis of cell samples may be done up to 3 days after fixation. Results indicate that the percentages of Class I, Class II, CD3, CD8, CD4, CD45, monocyte, gamma-delta T-cell populations, and total number of granulocytes identified using this method were comparable to standard values or to values obtained following separation of white blood cells from red blood cells. The percentage of labeled B-cells was lower than standard values. Total assay time from receipt of blood to acquisition of data by flow cytometry required less than 2 h. This modified assay was shown to be simple, reliable, and useful for screening large numbers of porcine samples in a minimal period of time.

Quantitative relationship of systemic virus concentration on growth and immune response in pigs.

Ninety-six pigs from a herd naive for porcine reproductive and respiratory syndrome (PRRS) virus were weaned (10 +/- 3 d of age), penned individually in isolation rooms, and, at 29 +/- 4 d of age, oronasally inoculated with a 2-mL dose of 10(4.3) JA142 PRRS virus/ mL. Body weight; feed intake; and serum concentrations of PRRS virus, interferon, and alpha1-acylglycoprotein were determined for each pig every 4 d on d -8 to 24 postinoculation to quantify the effect of PRRS exposure on the immune response and growth of pigs. Another objective was to determine whether a quantitative relationship between a measure of systemic (serum) virus concentration and pig growth exists. Serum PRRS virus and interferon peaked at 10(5) virus/mL and 69% protection, respectively, at 4 d postinoculation and then declined steadily. Serum alpha1-acylglycoprotein concentration peaked at 12 d postinoculation. Pig weight gains and feed intake were reduced sharply in the initial 8 d postinoculation and to a lesser degree for 24 d postinoculation. The serum concentration of virus and to a lesser degree serum concentrations of interferon and alpha1-acylglycoprotein were quantitatively related to body weight gain and feed intake. The magnitude of the relationship was dependent on the stage of recovery from PRRS infection. Specifically, each log increase in serum virus concentration was associated with a reduction of 4-d pig gain and feed intake of .047 kg and .189 kg, respectively, in 5.5-kg pigs 4 d postinoculation and .085 kg and .036 kg, respectively, in 12.5-kg pigs at 20 d postinoculation. Based on these data, factors that minimize the systemic presence of a virus in pigs result in improvements in pig growth that are quantitatively related to the degree of systemic virus elimination or minimization.

Evaluation of 2-deoxy-D-glucose for induction of a stress response in pigs.

OBJECTIVE: To determine the most effective route and dose for 2-deoxy-D-glucose (2DG) administration in swine, kinetics of 2DG, endogenous glucose concentration in the blood, effects of 2DG on cortisol concentration, and effects of 2DG administration in vivo on lymphocyte proliferation in vitro. ANIMALS: 14 Salmonella-free male and female mixed-breed pigs. PROCEDURE: A cannula was inserted in the femoral artery of each pig to allow for frequent blood collection with minimal external stress. The concentration and duration of 2DG in the blood was monitored while varying dose (250, 500, or 750 mg/kg of body weight) and route (IV, SC, IM, or IP) of 2DG administration. Blood samples were collected at various time points and assayed for lymphocyte response to concanavalin A and cortisol, endogenous glucose, and 2DG concentrations. RESULTS: The 2 best routes for administration of 2DG were IV and SC. If the IV route was chosen, the optimal dose was 500 mg of 2DG/kg; the optimal dose for SC administration was 750 mg/kg. CONCLUSIONS: 2DG induces a stress response in pigs similar to that in rodents. The use of 2DG in a porcine stress model should be effective for studying the possible role of stress in the pathogenesis and shedding of microorganisms.

Cloning and expression of porcine recombinant soluble tumor necrosis factor receptor 1.

OBJECTIVE: To clone, sequence, and express porcine recombinant soluble tumor necrosis factor receptor 1 (sTNFR1). PROCEDURE: A polymerase chain reaction (PCR)-based library enrichment technique was used to isolate a fragment of porcine TNFR1. The mature extracellular domain of porcine TNFR1 was subcloned into an expression vector and expressed in Escherichia coli as a fusion protein. Protein product was purified by immunoaffinity chromatography, using a commercially available affinity gel specific for the marker peptide of the fusion protein. The bioactivity of the purified protein was tested for its ability to inhibit TNF-mediated cytotoxicity in a PK(15) bioassay. RESULTS: A 927-base pair fragment of porcine TNFR1 encoding the entire extracellular and transmembrane domains, as well as 75 amino acids of the cytoplasmic domain, was isolated from a porcine lung cDNA library. The extracellular domain was expressed as a soluble TNFR1 fusion protein with a yield of 120 to 150 microg/L of culture. Affinity-purified porcine sTNFR1 was able to inhibit TNF-mediated cytotoxicity of porcine PK(15) cells in dose-dependent manner. CONCLUSIONS: Porcine recombinant sTNFR1 inhibits TNF bioactivity in vitro. This recombinant protein will be useful for developing TNFR1 antibodies and studying the roles of TNF and TNFR1 in the pathogenesis of infectious diseases in swine.

Comparison of the PK(15)- and WEHI 164 (clone 13)-based bioassays for detection of porcine tumor necrosis factor.

OBJECTIVE: To determine relative sensitivities of the PK(15)- and WEHI 164(13)-based bioassays for detection of tumor necrosis factor alpha (TNF). SAMPLE POPULATION: Recombinant human, murine, and porcine INF, and serum from pigs given endotoxin IV. PROCEDURE: Two cell lines were used as targets for recombinant human, murine, and porcine TNF cytotoxicity bioassays. Pigs were given sublethal doses of endotoxin to obtain serum samples containing high activity of porcine TNF. Serum TNF activity was tested, using both cell lines. Viable cells were detected by addition of dimethylthiazol diphenyltetrazolium bromide after 18 to 20 hours' incubation with samples containing TNF. RESULTS: The 2 cell lines tested had different sensitivities to human, murine, and porcine TNF. Compared with WEHI 164(13) cells, PK(15) cells were 50 times less sensitive to murine TNF and 15 times less sensitive to human TNF. However, PK(15) cells were 4 times more sensitive to recombinant porcine TNF and 15 times more sensitive to porcine serum containing TNF. CONCLUSIONS: The PK(15) cell line was more sensitive to porcine TNF-mediated lysis than was the WEHI 164(13) cell line. The PK(15)-based TNF bioassay will be especially useful for study of infectious disease processes in swine, particularly where low activity of TNF exists.

Effect of dose on the immune response and persistence of Salmonella choleraesuis infection in swine.

OBJECTIVE: To establish the effect of dose on persistence of and immune response to Salmonella choleraesuis in swine. DESIGN: 19 Salmonella-free pigs were allotted to 4 groups. Groups 1 (n = 5), 2 (n = 5), and 3 (n = 5) were inoculated intranasally with 10(9), 10(6), and 10(3) colony-forming units of S choleraesuis, respectively. Group 4 (n = 4) served as uninoculated controls. PROCEDURE: Pigs were monitored for clinical signs of disease and bacterial shedding. Serum and lymphocytes were obtained to measure immune responses. Pigs from groups 1, 2, and 4 were necropsied at postinoculation (PI) weeks 6 and 15. Pigs from groups 3 and 4 were necropsied at PI weeks 6 and 10. RESULTS: Pigs in group 1 shed S choleraesuis through PI week 15 and were tissue positive at PI weeks 6 and 15. Pigs in group 2 were tissue positive for S choleraesuis until PI week 6 and continued shedding through PI week 9. Salmonella choleraesuis was not recovered at any time from pigs in groups 3 or 4. Pigs in groups 1, 2, and 3 had serum IgG and IgM titers to S choleraesuis lipopolysaccharide and soluble antigens. Pigs in all groups had a lymphocyte response to concanavalin A, and pigs in groups 1 and 2 had a lymphocyte response to S choleraesuis endotoxin. Pigs in group 1 had a lower stimulation index in response to both antigens, indicating some form of lymphocyte immunosuppression. CONCLUSIONS: Persistence of S choleraesuis in host tissues is dose dependent. Short-term persistence can occur after a dose as low as 10(6) colony-forming units of S choleraesuis. Higher doses result in development of long-term carrier status, which may be related to the observed lymphocyte immunosuppression.

Natural transmission of Salmonella choleraesuis in swine.

This experiment was designed to study the natural transmission of Salmonella choleraesuis in swine. Forty pigs were divided into three groups. Group 1 (n = 12) was challenged with 10(8) CFU of S. choleraesuis per ml by intranasal inoculation. One day postinoculation (p.i.), group 2 (n = 24) was commingled with group 1. Group 3 (n = 4) served as uninoculated controls. Serum samples were collected weekly. Blastogenesis assays and necropsies were performed at 1, 2, 4, 6, 9, and 12 weeks p.i., and 16 tissue samples per pig were collected and cultured. Environmental (pooled feces from the pen floor) levels of S. choleraesuis were 2.61 log10 CFU/g of feces at 24 h p.i. (immediately prior to commingling). Severe clinical signs were observed in groups 1 and 2. The results indicated that at least 16% of group 2 pigs were shedding S. choleraesuis within 24 h of commingling. At 1 week p.i., 32 of 32 group 1 and 39 of 62 group 2 tissue samples were positive for S. choleraesuis. Only 3 of 12 group 2 pigs were positive at 6, 9, and 12 weeks (1 pig for each week), indicating that only a small proportion of infected swine become long-term carriers. At 12 weeks p.i., only the colon and colonic lymph node samples of one pig from group 2 were positive. Humoral, mucosal, and cellular immune responses were similar between groups 1 and 2. These data demonstrate that a few pigs shedding low levels of Salmonella organisms before slaughter can result in rapid transmission and subsequent shedding by many swine.

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.

Tumor necrosis factor-alpha production in swine after oral or respiratory challenge exposure with live Salmonella typhimurium or Salmonella choleraesuis.

A series of experiments was conducted to document tumor necrosis factor-alpha (TNF) activity in serum of swine after inoculation with Salmonella spp endotoxin and after oral or respiratory tract challenge exposure with live Salmonella spp. For experiment 1, a potentially lethal dose of S typhimurium endotoxin (25 micrograms/kg of body weight) was administered IV, and serum TNF activity was measured. High TNF (approx 700 IU/ml) activity at 1 to 2 hours after administration of the inoculum was associated with death, whereas lower TNF (approx 30 IU/ml) activity was associated with a general prolonged state of shock. For experiment 2, pigs were administered a nonlethal dose (5 micrograms/kg, IV) of either S typhimurium or S choleraesuis endotoxin. Difference in the ability to induce porcine serum TNF activity was not observed between strains. During experiment 3, pigs were inoculated with 10(4) colony-forming units of S typhimurium chi 4232 either orally by gelatin capsule (GC) or by intranasal (IN) instillation. A late serum TNF response (17 IU/ml) was measured at 6 weeks after IN inoculation. A serum TNF response was not detected in GC-inoculated pigs. All tissues and feces were test-negative for S typhimurium prior to the 6-week TNF response. Serum TNF activity may be related to clearance of S typhimurium after respiratory tract exposure, but it is not important to or indicative of clearance of orally presented S typhimurium in swine. During experiment 4, pigs were inoculated with 10(6) colony-forming units of S typhimurium chi 4232 similarly as for experiment 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Alternate routes of invasion may affect pathogenesis of Salmonella typhimurium in swine.

Transmission of Salmonella typhimurium in swine is traditionally believed to occur by the fecal-oral route, with invasion through the intestinal wall and Peyer's patches. However, involvement of the upper respiratory tract may be equally important. An esophagotomy was performed on 6- to 8-week-old pigs. Esophagotomized pigs were challenged intranasally with 10(9) CFU of S. typhimurium cells and necropsied at 3, 6, 12, and 18 h postinoculation (p.i.). By 3 h p.i., S. typhimurium was recovered from cecum, colon, head, and thoracic tissues and from the middle ileum involving a large number of Peyer's patches. The ileocolic lymph nodes and ileocolic junction were not positive for S. typhimurium until 6 and 12 h p.i., respectively. Additional pigs were inoculated transthoracically with 10(9) CFU of S. typhimurium and necropsied at 3 and 18 h p.i. By 3 h p.i., all tissues were positive for S. typhimurium. Tonsil explants seeded with 10(9) CFU of S. typhimurium indicated that within 6 h p.i., S. typhimurium was located within the tonsilar crypts. These data show that after intranasal inoculation, S. typhimurium rapidly appears in the gut tissues and suggest that the tonsils and lung may be important sites for invasion and dissemination of Salmonella species.

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.

Brucella abortus catalase is a periplasmic protein lacking a standard signal sequence.

A periplasmic catalase has been purified and cloned from Brucella abortus. The functional enzyme is a tetramer with a subunit molecular weight of 55,000. All evidence indicates that a typical N-terminal signal sequence is not associated with the export of this protein to the periplasm.