Coli J5 Research Articles

Effects of Adjuvants on Safety and Efficacy of an Escherichia coli J5 Bacterin

The effects of using a water-soluble adjuvant or an emulsified oil-based adjuvant on the safety, antibody titer, and clinical responses of an Escherichia coli J5 bacterin were tested in an experimental infection trial. Fifty-one cows were assigned to 17 blocks of 3. Two cows within each block of 3 were vaccinated with a commercially prepared E. coli J5 bacterin containing either a water-soluble adjuvant or the same bacterin preparation emulsified in oil. One cow in each block was an unvaccinated control. Cows were immunized at drying off and 42 d later. The right or left front mammary quarter of each experimental cow was challenged by intramammary infusion of E. coli 727 between 14 and 35 DIM. Areas of inflammation at the primary injection site were greater 1, 2, and 3 d following primary vaccination for bacterin containing oil-in-water adjuvant compared with bacterin containing water-soluble adjuvant. Whey anti-E. coli J5 IgG titers were higher at calving for cows vaccinated with bacterin containing oil-in-water adjuvant than for cows either vaccinated with bacterin containing water-soluble adjuvant or unvaccinated controls. Serum x-E. coli J5 IgG titers were higher at calving for vaccinated cows than for unvaccinated controls. Peak bacterial counts in milk from challenged quarters were greater for unvaccinated controls than for cows vaccinated with bacterin containing water-in-oil adjuvant. Bacterial counts in milk from challenged quarters and clinical score both were greater in unvaccinated controls than cows vaccinated with bacterin containing water-in-oil adjuvant between 12 and 24h postchallenge. Clinical responses were similar between unvaccinated controls and cows vaccinated with bacterin containing water-soluble adjuvant.

Growth responses of Escherichia coli to immunoglobulin G from cows immunized with ferric citrate receptor, FecA.

Effects of purified immunoglobulin (Ig) G from cows immunized with ferric citrate receptor, FecA, on the in vitro growth of Escherichia coli were investigated. Twenty-one cows were assigned to one of 3 treatments: 1) FecA immunization, 2) E. coli J5 bacterin immunization, and 3) unimmunized control. FecA was derived from E. coli UT5600/pSV66. Immunoglobulin G was purified from pooled colostral whey for each treatment group. The IgG from FecA immunized cows had higher titers against FecA compared with other treatment groups. Bacterial isolates tested were 14 E. coli from intramammary infections and E. coli UT5600/pSV66. Iron depletion decreased the growth of E. coli compared with growth in Fe-replete medium. The presence of IgG further decreased the growth compared with the growth under iron restriction alone. Bacterial growth did not differ among IgG sources nor between IgG concentrations. Replenishing media with exogenous iron overrode the inhibitory effects of the Fe-depletion and IgG. Vaccinating cows with FecA had little effect on the growth inhibitory properties of IgG toward E. coli mastitis isolates cultured in Fe-deplete media.

Opsonic Activity of Serum and Whey from Cows Immunized with the Ferric Citrate Receptor

The effects of immunizing dairy cows with the ferric citrate receptor, FecA, on the opsonic activity of serum and whey were measured in a phagocytosis assay. Fifteen cows were assigned to five blocks of three cows based on date of expected parturition. Cows within a block were randomly assigned to one of three treatments: 1) FecA immunization, 2) immunization with a commercially available Escherichia coli J5 bacterin, and 3) unimmunized controls. Cows were challenged at approximately 21 DIM by intramammary infusion of E. coli 727 into one mammary quarter. Escherichia coli 727 were opsonized for the phagocytosis assay with either 10% heat-inactivated serum or 50% heat-inactivated whey collected from each cow at calving, immediately before challenge and 7 d after challenge. Cows immunized with FecA or the E. coli J5 bacterin had increased IgG titers against FecA and E. coli 727 compared with unimmunized control cows. However, sera and whey collected from cows immunized with FecA did not enhance opsonization of E. coli 727 compared with sera and whey from control cows. Immunization with the E. coli J5 bacterin increased opsonization of sera greater than immunization with FecA. Immunoglobulin M antibody titer against E. coli 727 in whey and phagocytic indexes were positively correlated. The phagocytic index of whey immediately before challenge and 7 d after challenge were negatively associated with peak bacterial counts in mammary quarters challenged with E. coli 727. Results of the current trial suggest that the immune response resulting from immunization with FecA did not enhance opsonization and in vitro phagocytosis of E. coli 727.

Evaluation of commercially available Escherichia coli J5 bacterin as protection against experimental challenge with Pasteurella multocida in rabbits

Abstract Objective To evaluate the ability of commercially available Escherichia coli J5 bacterin to protect rabbits from experimental challenge with Pasteurella multocida. Animals 40 P multocida-free New Zealand White rabbits. Procedures Rabbits were assigned to 1 of 4 groups of 10 rabbits each. Three of the groups were inoculated SC with J5 bacterin at 8 weeks old. Inoculation was repeated 3 and 6 weeks later. The fourth group was not inoculated and served as controls. Groups 1, 2, and 3 were given 109, 108, and 107 colony forming units (CFU), respectively. Response was monitored by titer assessment, using an E coli J5 antigen capture ELISA. Five weeks after the last inoculation, all rabbits were challenged with P multocida and observed for an additional 5 weeks. Clinical, hematologic, serologic, culture, and necropsy data were collected. Results Inoculation of rabbits with 109 CFU of E coli J5 bacterin-induced titers that were significantly greater than titers of rabbits vaccinated with 108 or 107 CFU or those in controls. The incidence of acute bacteremia was lower in rabbits with high titers. At necropsy, prevalence of lesions typical of P multocida was not significantly different among groups. Prevalence of histologic lesions was also not significantly different among groups. Conclusions and Clinical Relevance Although the bacterin induced considerable antibody response and possibly reduced the rate of bacteremia, antibodies were not protective against long-term colonization or infection of the frontal sinuses or tympanic bullae by the challenge strain of P multocida. This bacterin in its currently available form is unlikely to aid in reducing the prevalence of pasteurellosis in rabbits. (Am J Vet Res 1999;60:853–859)

Responses of Antibody Titers to Intramammary Immunization with Escherichia coli J5 Bacterin

The effect of an immunization schedule on responses of antibody titers was tested following vaccination with an Escherichia coli J5 bacterin. Eighteen cows were equally distributed among three immunization schedules: 1) subcutaneous injection at 14 d prior to the end of lactation, intramammary immunization at 7 d after drying off, and subcutaneous injection at 30 d into the dry period; 2) subcutaneous injections at drying off, at 30 d into the dry period, and within 12h after calving; and 3) unimmunized controls. The E. coli J5 bacterin consisted of 5ml of 10 9 boiled cells/ml of 0.9% NaCl plus 0.005% phenol emulsified with 5ml of Freund's incomplete adjuvant. Subcutaneous injections were administered on the upper part of the rib cage, posterior to the scapula. Intramammary immunizations of 2.5ml of bacterin were infused via the teat canal into each of the four mammary glands. Intramammary immunization increased rectal temperatures at 12h after infusion, but subcutaneous injections did not induce febrile responses. Intramammary immunization enhanced immunoglobulin G titers in serum and whey on d 0 of lactation compared with subcutaneous immunizations. Immunoglobulin G titers in serum also were greater at d 30 of the dry period and at d 14 and 21 of lactation for cows that received intramammary immunization than for cows that were vaccinated by subcutaneous injections only. Immunoglobulin M titers in whey and serum on d 21 of lactation were greater for cows that received intramammary immunizations than for cows that were immunized by subcutaneous injections only.

C5b-7 and C5b-8 precursors of the membrane attack complex (C5b-9) are effective killers of E. Coli J5 during serum incubation

The finding that C9-deficient sera (C9D) can kill serum sensitive strains of Gram-negative bacteria by us and other investigators, questions the role of C9 in the membrane attack complex as necessary for cell death. In these studies we have demonstrated that C5b-8 complexes generated on E. coli J5 during incubation in C9-depleted and C9-neutralized sera are effective in killing Gram-negative bacteria. In the same study, we extended our investigations to show that the deposition of C5b-7 complexes (from C8-deficient [C8D], C8 depleted and C8-neutralized sera) is also effective in killing Gram-negative bacteria. In all cases, these studies demonstrated that when E. coli J5 was incubated with C8D, C9D and pooled normal human serum [PNHS], deposited C5b-9 complexes from PNHS produced more killing than C5b-7 or C5b-8 complexes alone. These experiments clearly demonstrated that C5b-7 and C5b-8 complexes are bactericidal and that multimeric C9 within C5b-9 is not an absolute requirment for inner membrane damage and cell death of Gram-negative bacteria.

Effect of vaccination with an Escherichia coli bacterin-toxoid on milk production in dairy cattle

Objective To determine whether vaccination of lactating cattle with an Escherichia coli J5 bacterin-toxoid would produce a significant short-term change in milk production. Design Randomized, controlled clinical trial. Animals 84 healthy, lactating cows (42 Holsteins and 42 Jerseys). Procedure Control and vaccinated cows were paired on the basis of breed, days in milk, daily milk production 1 week prior to vaccination, and parity. One cow in each pair was inoculated IM with a commercially available bacterin-toxoid according to label directions; the other cow was given saline solution. Cows were milked twice daily for 5 days before and 5 days after inoculation. Milk production was compared by ANCOVA. Results Vaccinated cows produced significantly less milk than did control cows at the second and third milkings after inoculation. At these milkings, milk production in vaccinated cows was approximately 7% less than that of controls. Clinical Implications Vaccination of lactating cattle with an E coli J5 bacterin-toxoid may cause a significant short-term decrease in milk production. (J Am Vet Med Assoc 1996;209:1291–1293)

The effect of Escherichia coli J5 and modified live Salmonella dublin vaccines in artificially reared neonatal calves

One thousand neonatal calves, allocated in a factorial design into four groups, were vaccinated subcutaneously with two doses each of either killed Escherichia coli (O111:B4) J5 bacterin or a UC Davis modified live, genetically altered (aro −) Salmonella dublin vaccine, or both, or with a placebo. In this prospective double-blind study to determine the immunogenicity and protective effects of both vaccines on bovine neonates in field conditions, calves were observed daily until 2 months of age, and serum samples from selected study calves were obtained at five different time points. No clinical adverse vaccine reactions were observed. Overall mortality was 7.5% (75 of 1000), E. coli and S. dublin infection being the most commonly associated aetiological agents of deaths. Both J5 ( p<0.01) and Salmonella ( p=0.05) vaccines were significantly effective in reducing the mortality rate but without an additive effect. The role of passive transfer was important in calf survival. The E. coli J5 and (aro −) S. dublin vaccination schedule employed significantly ( p<0.001) elevated J5 and Salmonella-specific serum ELISA antibody titres, respectively, by the sixth week of age.

Passive immunotherapy in neonatal calves—I. Safety and potency of a J5 Escherichia coli hyperimmune plasma in neonatal calves

A pair of studies designed to assess the clinical safety and potency of hyperimmune plasma administration was undertaken in neonatal calves. Bovine plasma from Holstein heifers hyperimmunized with a mutant Escherichia coli O111:B4 (J5) vaccine which had a geometric mean (GM) immunoglobulin G (IgG) ELISA titer of 3.5 × 10 4, was administered subcutaneously to ≤2-day-old calves. In the first (study A) of two prospective trials, hyperimmune plasma was administered in two doses to colostrum-deprived (CD) ( n=7) and colostrum-fed (CF) ( n=16) neonatal dairy calves. Data were collected immediately before (0 h) and 6, 24, 48, 72, 96, 120 and 288 h after the first plasma administration. Total serum protein and serum IgG concentration elevated in both groups with a significant ( p<0.01) rise of E. coli J5-specific IgG from the corresponding baseline by 6 h post-plasma administration. In the second (study B) trial, calves ( N=75) allocated into three protocol groups received two doses of either the hyperimmune (TT) J5 plasma ( n=30) or the control (CT) plasma ( n=30), and the third group (NT) received no plasma ( n=15). Data were collected up to 96 h post-plasma administration. Serum IgG and J5 specific ELISA antibody titer increased significantly ( p<0.01) in TT calves compared to the other group calves. The endotoxin contents of the administered plasma were ≤1 EU ml −1 by limulus amebocyte lysate (LAL) method and the collected physiological and hematological data values were similar in all groups in both trials. In addition, no immediate adverse reaction or death was observed in any phase of the plasma administration. The E. coli J5 hyperimmune bovine plasma, as prepared and administered to neonatal calves in the current trials, proved to be a safe and potent biomedical fluid.

118 TREATMENT OF SEVERE INFECTIOUS PURPURA IN CHILDREN WILL HUMAN PLASMA FROM DONORS IMMUNIZED WITH ESCHERICHILA COLI JS: A PROSPECTIVE, DOUBLE BLIND STUDY

Immunotherapy with serum obtained from immunized volunteers with Escherichia Coli J5 vaccine was shown to decrease the mortality from Gram-negative shock. We analyzed the effect of anti-J5 plasma on the course and mortality of severe infectious purpura. Patients with a clinical diagnosis of sepsis wiwth purpuric lesions were enrolled if they were in shock. They received either anti-J5 plasma or control plasma. Before the administration of the plasma, 6 hours after and after 5 days of evolution, serum levels of anti-J5 antibodies, tumor necrosis factor alpha (TNF), interleukin-6 (IL-6) and elastase-al-antiprotease complexes were determined. 73 patients were randomized, 40 in the anti-J5 and 33 in the control group. Clinical and biological risk factors were similar in the two groups. However, TNF serum concentrations were 974±173pg/ml versus 473±85pg/ml, (p=0.023) and IL-6 serum concentrations were 129±45 versus 19±5ng/ml, (p=0.005) in the control group and in the treated group respectively. The duration of hypotension, of vasopressor therapy and of respiratory assistance and the occurrence of systemic complications were similar in the 2 groups. The mortality rate was 36 % in the control group and 25% in the treated group (p=0.317), (risk ratio:0.58;95% confidence interval:0.19-1.79). This trend disappeared after correction for imbalances at randomization using a logistic regression model. Anti-J5 plasma did not change the course and the mortality of severe infectious purpura in children.

Effect of immunization with Escherichia coli J5 on graft-versus-host disease induced by minor histocompatibility antigens in mice.

We have used a cell-wall-deficient mutant of Escherichia coli (E coli J5) to study the effect of active and passive immunization against bacterial endotoxin (ET) in a murine model of acute graft-versus-host disease (GVHD) induced by minor histocompatibility antigens. C57BL/B6 (H-2b) mice were irradiated and grafted with 1 X 10(7) anti-Thy-1.2 and complement-treated bone marrow cells and 5 X 10(7) spleen cells from LP/J (H-2b) donors. Groups of mice were immunized against J5--either actively immunized with killed J5 cells or pure J5 lipopolysaccharide or passively immunized with rabbit anti-J5 antiserum (R alpha J5). Controls included irradiation controls, negative controls (conventional mice grafted with depleted bone marrow only), positive controls (conventional mice grafted with bone marrow and spleen cells), and mice passively immunized with normal rabbit serum (NRS). Positive control mice developed GVHD and all died by day +77. Active immunization partially protected against the rapid weight loss due to GVHD (P less than 0.05) but mice had an earlier onset of GVHD (P less than 0.05) and a shortened survival after onset (P less than 0.05). Median survival time (MST) and overall survival were unchanged. Passive immunization with R alpha J5 delayed death (P less than 0.01), increased MST (P = 0.01), increased overall survival (P less than 0.01), and protected against weight loss (P = 0.01). NRS had no beneficial effect.

Influence of endotoxin on graft-versus-host disease after bone marrow transplantation across major histocompatibility barriers in mice.

Much clinical and experimental data suggest that infection and graft-versus-host disease (GVHD) are intimately associated, and that bacterial endotoxin (ET), a potent immunostimulant, influences the severity of GVHD. We have used a cell-wall-deficient mutant of Escherichia coli (E coli J5) to study the effect of active and passive immunization against ET in a murine model of GVHD induced by major histocompatibility antigens. CBA/Ca (H-2k) mice were irradiated and grafted with 1 X 10(7) bone marrow cells from C57BL/B6 (H-2b) donors. Groups of mice were immunized against J5: either actively immunized with killed J5 cells or pure J5 lipopolysaccharide, or passively immunized with rabbit anti-J5 antiserum (R alpha J5). Controls included irradiation controls, negative controls (syngeneic graft), positive controls (conventional mice receiving allogeneic graft), mice immunized with normal rabbit serum, Freund's adjuvant (FA), or human serum albumin (HSA) in FA. Active immunization with J5 exacerbated the effects of GVHD as indicated by increased weight loss (P = 0.002) and earlier death (P = 0.043). In contrast, immunization with HSA protected against weight loss (P = 0.028), and improved survival (P = 0.008). Passive immunization with J5 had no effect. These observations support the hypothesis that ET influences the pathogenesis of GVHD, and provide a useful model for studying the effects of ET in a well-defined immunological system.

ANTIBODY TITRES TO A ROUGH-MUTANT STRAIN OF ESCHERICHIA COLI IN PATIENTS UNDERGOING ALLOGENEIC BONE-MARROW TRANSPLANTATION: Evidence of a Protective Effect Against Graft-versus-host Disease

Much clinical and experimental evidence suggests that infection and graft-versus-host disease (GvHD) are commonly associated as complications of bone-marrow transplantation (BMT). A likely basis for this association is the gram-negative faecal flora, the origin of many septicaemias and a source of bacterial endotoxin, which has potent immunostimulatory effects. A rough-mutant strain, Escherichia coli J5, has only core determinants in its endotoxin, and antibodies to E coli J5 protect animals and human beings from the consequences of septic shock. Naturally occurring antibodies to E coli J5 ("anti-endotoxin") were assayed in serum from patients undergoing BMT, healthy controls, and patients with obstructive jaundice. BMT recipients had significantly lower titres than the other two groups. Furthermore, the titre of IgM class anti-J5 antibody was significantly associated with protection from GvHD.

Detection of antibodies against lipopolysaccharides of Escherichia coli and Salmonella R and S strains by immunoblotting.

Antisera raised against several smooth and rough strains of Escherichia coli and Salmonella typhimurium were tested against lipopolysaccharides (LPS) of homologous and heterologous strains. The LPS were separated by sodium dodecyl sulfate-gel electrophoresis, transferred to nitrocellulose paper, and overlaid with antisera. The results showed that antisera raised against smooth strains reacted with high- as well as low-molecular-weight bands of their corresponding LPS and showed very few cross-reactions. Anti-E. coli J5 antiserum cross-reacted with few strains in the core region. But, anti-S. typhimurium Ra antiserum cross-reacted with many more strains. When these sera were absorbed with either the homologous- or a heterologous-positive strain, reactions were abolished. It appears that reactions of anti-E. coli J5 antiserum and anti-S. typhimurium Ra antiserum with homologous and heterologous strains were not due to the same antibody. This immunoblotting technique proved to be a useful method to distinguish different antibodies in antiserum raised against LPS of gram-negative bacteria.

Prophylaxis of Pseudomonas aeruginosa infections in leukopenic mice by a combination of active and passive immunization.

Mice rendered leukopenic with cyclophosphamide and then challenged with viable Pseudomonas aeruginosa were used to determine the protective efficacy of active immunization against exotoxin A and of passive immunization with human antiserum to Escherichia coli J5, a rough mutant of Escherichia coli O111:B4. Neither treatment alone provided a greater degree of protection than its respective control. However, the combination of these treatments produced a moderate, yet consistent, increase in the survival of infected immunosuppressed mice.