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Summary

Development of age-dependent resistance to enterotoxigenic Escherichia coli was studied, using isolated enterocytes and brush border membranes (bbm) from 7-day-old and 7-week-old pigs. Binding of 125I-labeled heat-stable (125I-STa) enterotoxin to enterocytes and bbm was specific, temperature- and time-dependent, saturable, and partially reversible. Scatchard analysis revealed a single class of receptors. Mean ± sd avidity of binding (apparent affinity constant, Ka) of 125I-STa to enterocytes from 7-day-old and 7-week-old pigs was 2.14 ± 0.29 × 108 and 2.72 ± 0.25 × 108 L/mol, respectively. Numbers of STa receptors were calcuated to be 64,903 ± 2,900/enterocyte for 7-day-old pigs and 53,029 ± 3,117/enterocyte for 7-week-old pigs. Numbers of STa receptors expressed per milligram of bbm protein from 7-day-old pigs were 2.66 × 1011, compared with 2.29 × 1011 for bbm from 7-week-old pigs. By 5 minutes after addition of STa to reaction mixtures, intracellular cyclic guanosine monophosphate concentration increased 13.9-fold in enterocytes from 7-day-old pigs and 8.7-fold in enterocytes from 7-week-old pigs. The particulate guanylate cyclase activity associated with bbm from 7-week-old pigs was slightly more sensitive to low amounts of STa, compared with bbm from 7-day-old pigs; however, differences were not observed at intermediate and high amounts. These data indicate that lack of a secretory response to STa by older pigs is not attributable either to decreased numbers of STa receptors or to decreased signal response between the STa receptor and membrane-bound guanylate cyclase. Development of age-dependent resistance by porcine small intestine to STa appears to be attributable to steps in the secretory pathway that respond to increased concentration of cyclic guanosine monophosphate.

Free access
in American Journal of Veterinary Research

SUMMARY

Corynebacterium pseudotuberculosis phospholipase D (PLD) significantly affected viability of ovine neutrophils after 24 hours' exposure. This effect was more marked in cells that ingested PLD emulsified in oil. Treatment of neutrophils with PLD significantly (P < 0.05) reduced the ability of these cells to migrate toward activated sheep serum. The PLD was not chemotactic, but it activated normal sheep serum, producing factors that were chemotactic for neutrophils.

Free access
in American Journal of Veterinary Research

SUMMARY

Sublethal irradiation of BALB/c mice 4 hours prior to inoculation with 5 × 104 virulent Brucella abortus, caused significant (P < 0.01) reductions in bacterial numbers in comparison with numbers in unirradiated controls. Numbers of brucellae in the spleen were significantly lower by 5 days after inoculation and decreased thereafter, so that at 2 and 3 weeks after inoculation, there were up to 1,000-fold fewer organisms in the spleen of irradiated mice. The number of brucellae in the spleen increased in irradiated mice thereafter. The course of events in the liver was similar, but developed more slowly, and peak differences in bacterial numbers were about 1 log less. These phenomena were not attributable to differences in implantation of brucellae in the liver or spleen, nor to an abnormal distribution of organisms in other organs of irradiated mice. Irradiation of mice during the plateau phase of infection also resulted in significant (P < 0.05) reductions in bacterial counts in the spleen during the succeeding 4 weeks. Macrophage activation in the spleen, measured by a Listeria monocytogenes-killing assay, was significantly (P < 0.01) increased by irradiation alone at 1 week after inoculation and at that time was significantly (P < 0.01) greater in B abortus -infected, irradiated mice than in B abortus-infected controls. Histologic, cytologic, and immunologic studies revealed that the decrease in numbers of organisms between 1 and 2 weeks after inoculation in irradiated mice occurred at a time when their immune response to B abortus was suppressed and when numbers of neutrophils and monocytes infiltrating the spleen were significantly (P < 0.01) diminished. The increase in numbers of B abortus in organs of irradiated mice that began after the third week coincided with recovery of the immune response and an increase in numbers of neutrophils and monocytes in the infected organs. The course of B abortus infection was not substantially altered during the first 11 days after inoculation in mice infected at the height of a profound monocytopenia and neutropenia induced by azathioprine, a drug that by itself failed to activate macrophages. We hypothesized that, in irradiated mice, a rapid radiation-induced activation of resident macrophages to a brucellacidal state was coupled with an absence of newly formed monocytes in which virulent strains of B abortus could establish persistent infection, and that as susceptible monocytes emerged in mice recovering from the effects of irradiation, chronic infection became established.

Free access
in American Journal of Veterinary Research

Summary

A murine IgM monoclonal antibody causing bacterial agglutination was used in an immunoaffinity procedure to purify a serotype 1-specific polysaccharide epitope from Pasteurella haemolytica. The P haemolytica serotype 1-specific antibody was precipitated from peritoneal ascitic fluid, dialyzed, and covalently attached to cyanogen bromide-activated Sepharose 4B beads. Retention of purified antibody activity and coupling efficiency were > 99% when evaluated by elisa, agglutination testing, and protein determination. Potassium thiocyanate was selected as an eluant on the basis of reversible dissociation of bacterial agglutination and was titrated for the lowest effective concentration. Immunobead activity was observed microscopically by immobilization of encapsulated P haemolytica serotype 1 and its reversible dissociation after elution with 0.4M potassium thiocyanate. Specificity of immobilization was visualized, using P haemolytica serotypes 2 and 5, which were not bound, and by blocking serotype-1 binding with homologous capsular material. Saline-extractable capsular material from P haemolytica serotype 1 was used as an antigen source. After elution of the serotype 1-specific polysaccharide epitope, the product was dialyzed and analyzed, using chemical and immunologic methods. The immunoaffinity product contained no detectable protein and greater than half the original hexosamine content. Using defined monoclonal antibodies in elisa, titration of the original capsular material and the immunoaffinity product revealed specific retention of lipopolysaccharide, a 10- to 30-kd polysaccharide antigen common to all P haemolytica and P multocida serotypes, and serotype 1-specific capsular polysaccharide, indicating possible epitope sharing among polysaccharide antigens of P haemolytica serotype 1.

Free access
in American Journal of Veterinary Research

SUMMARY

The purpose of this in vitro study was to determine whether Pasteurella haemolytica capsular extract (ce) damages bovine pulmonary endothelial cells (ec) directly or through neutrophil-mediated mechanisms. Chromium 51-labeled ec were treated with the following variables: ce (1, 10, and 100 ng of protein/ml), ce and bovine neutrophils (106 cells/well), and ce and polymyxin B (500 U/ml). Although only minimal damage to ec occurred by 5 hours after treatment, by 22 hours after treatment, the 10-ng and 100-ng ce dose produced severe damage to ec, as indicated by 51Cr release, cellular detachment, and loss of monolayer confluency. The component in the ce that was toxic to the ec was lipopolysaccharide, evidenced by effective neutralization of the toxic effect with polymyxin B. Neutrophils inhibited the ce-mediated ec toxicity and were activated, as indicated by shape change and adhesion to ec monolayers. We concluded that the lipopolysaccharide component of ce causes direct damage to ec, which can be attenuated by neutrophils and polymyxin B.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To evaluate in vitro effect of the major fraction of outer membrane proteins of Pasteurella multocida with porin-like activities on some biological functions of bovine neutrophils.

Animals

Neutrophils from 5 adult cattle.

Procedure

Variations in such biological processes as actin polymerization and chemotaxis and evaluation of hydrogen peroxide attributable to variable concentrations of P multocida were recorded and compared. Data were obtained, using the porin and lipopolysaccharide (LPS) isolated from a strain of P multocida cultivated in brain-heart infusion (BHI) broth. Various concentrations of porin and LPS were analyzed to evaluate changes in functional activation and microbicidal activity of bovine neutrophils.

Results

The 37.5-kd major polypeptide of the outer membrane of P multocida was isolated. Presence of this porin was significantly correlated with variations of some biological functions of bovine neutrophils. These immunocompetent cells had a concentration-dependent increase in actin polymerization and chemotactic activity. A concentration-dependent variation in the oxidative burst also was observed.

Conclusions

The porins of gram-negative bacteria affect several biological functions of cells involved in the immune response as well as in inflammation. Significant correlation of results of in vitro experiments also was identified between porin and LPS effect. Pretreatment of bovine neutrophils with various concentrations of porin always caused a concentration-dependent increase in examined biological activities. (Am J Vet Res 1998;59:1270–1274)

Free access
in American Journal of Veterinary Research

Summary

Cutaneous reactivity to brucellin was evaluated in 10-month-old heifers vaccinated with low-virulence mutant strains of Brucella abortus and was compared with brucellin reactions in postparturient cows with active brucellosis. In the cows, the cutaneous lesion was characterized microscopically as severe, acute, serofibrinous vasculitis; dermal lesions at 6, 12, 25, and 48 hours after brucellin injection consisted of endothelial activation and perivascular exudation that led to progressive accumulation of fibrin, monocytes, macrophages, and lymphocytes. In vaccinated heifers, cutaneous tests were done, using standard brucellin, brucellin prepared from strain RB51, and the purified brucellar proteins-31K and superoxide dismutase. Negative-control cattle given saline solution, did not have cutaneous reactions. Standard brucellin induced the most marked reactions in vaccinated heifers. Brucellin from rough strain RB51 caused positive reactions in heifers vaccinated with strain 19, but reactions were variable in other groups. Skin lesions induced by purified superoxide dismutase and 31-kd proteins in vaccinated cattle were not acceptable for diagnosis. Marked variability of test responses in vaccinated cattle precludes field use of this test to determine vaccination status.

Free access
in American Journal of Veterinary Research

SUMMARY

Bovine pulmonary artery endothelial cells (bpaec) were labeled with 3H-arachidonic acid. Exposure of the labeled bpaec to Pasteurella haemolytica lipopolysaccharide (lps) resulted in a time- and dose-dependent release of radioactivity. The release was inhibited by 5 mM indomethacin, but inhibition was not caused by ≤ 500 μM indomethacin or hydrocortisone, which suggests that the release was caused primarily by a mechanism other than cyclooxygenase or phospholipase A2 metabolism of arachidonic acid.

Pasteurella haemolytica lps also caused increased adherence of bovine neutrophils to bpaec through independent effects on both cell types. The increased adherence was inhibited by treatment of either cell type with cycloheximide or actinomycin D prior to lps exposure, indicating that de novo protein synthesis was required in both cell types to promote the lps-induced adherence. Lipopolysaccharide may be an important factor in neutrophil-mediated effects in pneumonic pasteurellosis by causing increased neutrophil adherence and, thus, the vascular sequestration of neutrophils.

Together, these experiments provide additional evidence for the involvement of lps in pneumonic pasteurellosis. Moreover, they provide evidence of lps-induced endothelial activation, which could have broad ramifications in the inflammatory and immune responses of pneumonic pasteurellosis.

Free access
in American Journal of Veterinary Research

assay. Activation was performed at 95°C for 12 minutes. Twenty-five cycles of denaturation (94°C for 30 seconds), annealing (63°C for 30 seconds), and extension (68°C for 3 minutes) were performed. A final extension was performed at 72°C for 10 minutes

Full access
in American Journal of Veterinary Research

: effects on NKKB, activator protein-1, c-Jun N-terminal protein kinase, and apoptosis . J Immunol 2000 ; 165 : 5962 – 5969 . 10.4049/jimmunol.165.10.5962 7. Li EK Tam LS Tomlinson B . Leflunomide in the treatment of rheumatoid arthritis

Full access
in American Journal of Veterinary Research