Differential gene expression of the toll-like receptor-4 cascade and neutrophil function in early- and mid-lactating dairy cows.

During early lactation, neutrophils display several reduced immune functions. Particularly, a delayed recruitment of neutrophils into the infected udder seems to be one of the underlying events involved in the severity of postpartum Escherichia coli intramammary infections. The purpose of this study was to analyze the effect of in vitro chemotaxis and diapedesis on the expression of toll-like receptor-4 (TLR4)-related genes in bovine blood neutrophils isolated from 10 early-lactating (EL) and 10 mid-lactating (ML) cows. Functional characterization of the neutrophil population was performed by measuring phagocytosis and production of reactive oxygen species (chemiluminescence). Messenger RNA was extracted from neutrophils, and the expression of TLR4 and associated genes in EL and ML cows was analyzed by reverse-transcription quantitative PCR. To study the effect of chemotaxis and diapedesis on the expression of genes of the TLR4 cascade, neutrophils were stimulated to (trans)migrate in response to C5a using in vitro models. Our salient findings were that both neutrophil migration in vitro and lactation stage induced significant changes in the expression of several genes of the TLR4 signaling cascade. Before migration, expression of TRAF6, ATF3, RELA, IL8, and C5aR were lower in EL than in ML cows. Diapedesis and chemotaxis induced an increase in expression of TLR4, ATF3, and IL8 in both EL and ML cows. Diapedesis resulted in a downregulation of Syk, a TLR4-associated gene, in ML cows. This study shows that the perturbations in neutrophil functions during EL are accompanied by modulation of TLR4 pathway genes. These data can contribute to the understanding of the mechanisms explaining the relationship between stage of lactation and risk of severe E. coli mastitis.

Anaphylatoxin C5a-induced toll-like receptor 4 signaling in bovine neutrophils.

It is well known that signaling in neutrophils through both the complement component 5a (C5a) and C5a receptor (C5aR) and the toll-like receptor 4 (TLR4) pathways plays an essential role in innate defense. Neutrophil dysfunction, as seen during sepsis in severe mastitis during the periparturient period, is correlated with elevated concentrations of anaphylatoxin C5a. The aim of the current study was to elucidate the effect of C5a on TLR4 signaling in bovine neutrophils. Neutrophils were incubated with a high (but physiological) dose of purified C5a, and mRNA was extracted from neutrophils at different time points postincubation (PI). The incubation with C5a resulted in a biphasic C5aR expression profile, a phenomenon that might be explained by internalization (at 10 min PI) with subsequent reconstitution (starting at 40 min PI) of this receptor. The expression of TLR4, as well as its coreceptor, CD14, showed a similar biphasic change as observed with C5aR. In addition, changes in the mRNA expression levels of several genes belonging to the TLR4 pathway, such as TICAM-1, IKKα, and MAP3K7 were noted. The maximal expression of TLR4, CD14, and C5aR mRNA at 80 min PI was accompanied by a peak in IL8 mRNA, indicating that C5a is able to induce IL-8 production in neutrophils in vitro without the need of a costimulatory factor such as lipopolysaccharide. Moreover, a relatively constant expression of RELA was accompanied by increased expression of ATF3, an endogenous inhibitor of nuclear factor-κB mediated transcription, implying that C5a regulates TLR4 signaling and IL-8 synthesis independently. A significant time-dependent correlation was found between C5aR and TLR4, with the majority of the selected TLR4-dependent genes showing a significant correlation with C5aR at 80 min PI, when C5aR and TLR4 mRNA expression reached its maximum, suggesting crosstalk between both receptors. Taken together, this study showed that C5a is able to (1) alter the expression of genes belonging to the TLR4 pathway and (2) induce IL8 gene expression in bovine neutrophils. In addition, indications for cross-talk between complement activation and TLR4 signaling were found in the present study.

A novel splice variant of the Excitatory Amino Acid Transporter 5: Cloning, immunolocalization and functional characterization of hEAAT5v in human retina.

Excitatory Amino Acid Transporter 5 (EAAT5) is abundantly expressed by retinal photoreceptors and bipolar cells, where it acts as a slow glutamate transporter and a glutamate-gated chloride channel. The chloride conductance is large enough for EAAT5 to serve as an "inhibitory" glutamate receptor. Our recent work in rodents has shown that EAAT5 is differentially spliced and exists in many variant forms. The chief aim of the present study was to examine whether EAAT5 is also alternately spliced in human retina and, if so, what significance this might have for retinal function in health and disease. Retinal tissues from human donor eyes were used in RT-PCR to amplify the entire coding region of EAAT5. Amplicons of differing sizes were sub-cloned and analysis of sequenced data revealed the identification of wild-type human EAAT5 (hEAAT5) and an abundant alternately spliced form, referred to as hEAAT5v, where the open reading frame is expanded by insertion of an additional exon. hEAAT5v encodes a protein of 619 amino acids and when expressed in COS7 cells, the protein functioned as a glutamate transporter. We raised antibodies that selectively recognized the hEAAT5v protein and have performed immunocytochemistry to demonstrate expression in photoreceptors in human retina. We noted that in retinas afflicted by dry aged-related macular degeneration (AMD), there was a loss of hEAAT5v from the lesioned area and from photoreceptors adjacent to the lesion. We conclude that hEAAT5v protein expression may be perturbed in peri-lesional areas of AMD-afflicted retinas that do not otherwise exhibit evidence of damage. The loss of hEAAT5v could, therefore, represent an early pathological change in the development of AMD and might be involved in its aetiology.

Comparative analysis of using MTT and XTT in colorimetric assays for quantitating bovine neutrophil bactericidal activity.

Two different tetrazolium compounds were compared for use in a colorimetric assay for quantitating bovine neutrophil bactericidal activity against Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, and Brucella abortus. The tetrazolium compounds tested included 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sodium 3,3'-[1[(phenylamino)carbonyl]-3,4- tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT). The MTT and XTT colorimetric bactericidal assays were conducted by incubating antibody-opsonized bacteria with neutrophils in microtiter plates for 30 and 60 min at ratios of ten and 100 bacteria per neutrophil. Neutrophils were then lysed with saponin and samples were incubated 30 min with MTT or XTT plus coenzyme Q (CQ). Dead bacteria and lysed neutrophils did not react with MTT or XTT plus CQ. Live bacteria converted XTT to water soluble orange formazan in the presence of CQ and MTT to insoluble purple formazan. Absorption of formazan produced by bacteria from XTT was measured at 450 nm. Formazan produced by bacteria from MTT was solubilized by adding isopropanol and measured by absorption at 560 nm. Absorption of both types of formazan was directly related to viable bacteria cell number and used to determine the number of bacteria not killed by neutrophils. The percentage of bacteria killed by neutrophils was determined by extrapolation from a standard formazan curve that was derived by incubating MTT or XTT plus CQ with known numbers of bacteria. The XTT and MTT colorimetric bactericidal assays produced comparable results when used to measure bovine neutrophil bactericidal activity against S. aureus, E. coli, L. monocytogenes, and B. abortus. However, the assay using XTT was quicker and easier to perform because bacteria converted XTT to a formazan that did not need to be solubilized before measuring absorption.

Effect of Brucella abortus transfer factor in preventing murine brucellosis.

Mice vaccinated with a protein extract of attenuated Brucella abortus strain 19 had increased resistance to infection with virulent B. abortus strain 2308 and had increased antibody responses to strain 2308. However, resistance to infection and antibody responses were not increased when nonvaccinated recipient mice were given transfer factor preparations that were obtained from either vaccinated donor mice or strain 2308-infected donor mice. Vaccination of mice with the strain 19 extract plus treatment with each transfer factor preparation also did not further increase resistance to infection or antibody responses when compared with mice that received the vaccine alone. These results suggest that transfer factor from mice that have either vaccine-induced protective immunity to B. abortus or active B. abortus infections does not enhance antibody responses and resistance to infection with B. abortus.

The use of listeriolysin O in an ELISA, a skin test and a lymphocyte blastogenesis assay on sheep experimentally infected with Listeria monocytogenes, Listeria ivanovii or Listeria innocua.

Purified listeriolysin O (LLO) was evaluated as a specific antigen to detect both humoral and cell mediated immune responses of sheep infected with Listeria monocytogenes. Six sheep (two in each group) were orally inoculated with 10(10) organisms of L. monocytogenes, L. ivanovii, or L. innocua. Only the L. monocytogenes inoculated sheep had an elevated temperature (> 42 degrees C) and after 15 days had anti-LLO antibodies as assessed by an ELISA. In a blastogenesis assay, only peripheral blood mononuclear cells (PBMC) from L. monocytogenes-infected sheep responded to LLO, while PBMC from all the sheep responded somewhat to heat-killed L. monocytogenes bacteria. In a skin test, only L. monocytogenes-infected sheep exhibited a positive reaction to injected LLO, while all the Listeria-infected sheep reacted to heat-killed bacteria. On day 120 postinfection, all of the sheep were orally inoculated with L. monocytogenes. Only the four that had not been previously given L. monocytogenes exhibited an elevated temperature (> 42 degrees C). 80 days later, sera from all of the animals were positive for anti-LLO antibodies. Thus, prior exposure to L. ivanovii or L. innocua does not protect against a L. monocytogenes challenge. These results suggest LLO is an excellent antigen for use in detecting Listeria infection in sheep. However, whether LLO will be useful in differentiating chronically infected animals from animals that have recovered, has yet to be investigated.

Immune responses to superoxide dismutase and synthetic peptides of superoxide dismutase in cattle vaccinated with Brucella abortus strain 19 or RB51.

Antibody and lymph node cell-mediated immune responses to recombinant Brucella abortus strain 19 Cu-Zn superoxide dismutase (rSOD) and to three synthetic strain 19 Cu-Zn SOD peptides were measured during 2 to 12 weeks following vaccination of cattle with B. abortus strain 19 or RB51. Cattle vaccinated with strain 19 or RB51 did not produce antibody to rSOD and to the SOD peptides. Lymph node cells from cattle vaccinated with strain 19, but not with strain RB51, proliferated when incubated with either rSOD or one of the three tested SOD peptides (GGDNYSDKPEPLGG). These results suggest that neither the strain 19 nor the strain RB51 vaccine induces antibody production to SOD and only the strain 19 vaccine induces lymph node cell-mediated immune responses to SOD.

Comparative effects of bovine cytokines on cattle and bison peripheral blood mononuclear cell proliferation.

Proliferation of peripheral blood mononuclear cells (PBMC) from cattle and bison was measured following stimulation of PBMC with bovine cytokines. Bovine interleukin 1 beta (BoIL-1 beta), interleukin 2 (BoIL-2) or granulocyte-macrophage colony-stimulating factor (BoGM-CSF) at 0.1-100 U/ml were incubated for 48 h with PBMC alone or with PBMC and various mitogens. These included concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) or Escherichia coli 055:B5 lipopolysaccharide (LPS) at 10-0.1 micrograms/ml. BoIL-2 alone, but not BoIL-1 beta and BoGM-CSF alone, induced proliferation of cattle and bison PBMC in the absence of mitogens. In addition, BoIL-1 beta and BoIL-2, but not BoGM-CSF, enhanced proliferation of cattle and bison PBMC induced by mitogens. These results indicate that BoIL-1 beta and BoIL-2 stimulate cattle and bison PBMC proliferation in a similar manner, whereas BoGM-CSF does not appear capable of stimulating either cattle or bison PBMC proliferation.

Role of immune responses to a GroEL heat shock protein in preventing brucellosis in mice vaccinated with Brucella abortus strain RB51.

Resistance to infection with virulent Brucella abortus strain 2308 and antibody and lymphocyte proliferative responses to a recombinant 60 kDa B. abortus GroEL heat shock protein were measured in mice vaccinated with attenuated B. abortus strain RB51. Mice at 12-20 weeks after vaccination with 5 x 10(8) colony forming units (CFU) of strain RB51 had increased resistance to infection with strain 2308 and increased antibody and lymphocyte proliferative responses to GroEL following challenge infection with 2308. However, these mice at 12-20 weeks after vaccination did not have greater resistance to infection than mice vaccinated with 5 x 10(6) CFU of strain RB51, which had no increased antibody or lymphocyte proliferative response to GroEL. These results indicate that mice vaccinated with strain RB51 can have antibody and cell-mediated immune responses to GroEL during infection with virulent strain 2308, although neither response appeared to have an essential role in vaccine-induced immunity to brucellosis.

In vitro effects of live and killed Brucella abortus on bovine cytokine and prostaglandin E2 production.

Live and gamma-irradiated-killed Brucella abortus strain 2308 increased interleukin 1 (IL-1), but not interleukin 2 (IL-2), interferon-gamma (IFN-gamma), or prostaglandin E2 (PGE2) production when incubated with normal bovine peripheral blood mononuclear cells (PBMC). Live B. abortus was more effective than killed B. abortus in stimulating IL-1 production by normal PBMC. Both live and killed B. abortus were equally effective in suppressing IL-2 and IFN-gamma production by Concanavalin A-stimulated PBMC. Incubation of PBMC with the cyclo-oxygenase inhibitor, indomethacin, blocked PGE2 synthesis, but did not further enhance IL-1 production or prevent suppressed IL-2 and IFN-gamma production that was induced by live and killed B. abortus. These results suggest that B. abortus-induced suppression of IL-2 and IFN-gamma production did not appear to be mediated by the suppressive prostaglandin, PGE2, or other cyclo-oxygenase metabolites.

A colorimetric assay for quantitating bovine neutrophil bactericidal activity.

A colorimetric assay was developed for quantitating bovine neutrophil bactericidal activity against Staphylococcus aureus. The procedure used the tetrazolium compound, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The assay was conducted by incubating antibody-opsonized S. aureus with neutrophils in microtiter plates for 1 h at a ratio of 10 bacteria per neutrophil. Neutrophils were then lysed with saponin. The MTT was added and samples were incubated for 10 min. Live S. aureus reduced MTT to purple formazan. Dead bacteria and lysed neutrophils did not react with MTT. Bacterially-reduced formazan was solubilized by adding isopropanol and formazan production was quantitated by measuring absorption at 560 nm. Absorption of formazan was directly related to viable bacteria cell number and was used to determine the number of S. aureus not killed by neutrophils. The percentage of bacteria killed by neutrophils was determined by extrapolation from a standard formazan curve that was derived by incubating MTT with known numbers of S. aureus. The colorimetric MTT assay detected suppressed bactericidal activity after in vitro treatment of bovine neutrophils with colchicine, cytochalasin B, or phorbol 12-myristate 13-acetate. In vitro treatment of neutrophils with low levels of recombinant bovine interferon gamma (rBoIFN-gamma) enhanced bactericidal activity, whereas high levels decreased activity. These results suggest the colorimetric MTT bactericidal assay is efficacious in detecting modulation of bovine neutrophil bactericidal activity. Furthermore, the MTT assay has many advantages over traditional bactericidal assays in that it is sensitive, inexpensive, requires less than 3 h to complete, and can analyze many neutrophil samples in a single day.

Comparative analysis of immune responses in cattle vaccinated with Brucella abortus strain 19 or strain RB51.

Immune responses were measured for 12 weeks following vaccination of cattle with either Brucella abortus strain (S) 19 or SRB51. Cattle vaccinated with S19, but not with SRB51, produced antibodies that agglutinated B. abortus S1119 in the standard tube agglutination test. Cattle vaccinated with S19 or SRB51 produced antibodies to the surface antigens of SRB51 when measured by a dot enzyme-linked immunosorbent assay. Superficial cervical lymph node (LN) cells obtained by biopsy at 10 and 12 weeks from cattle given the S19 or SRB51 vaccine exhibited similar proliferative responses when incubated in vitro with gamma-irradiated B. abortus S2308. At 10 and 12 weeks after vaccination, LN cells obtained from cattle given S19 or SRB51 proliferated to 22 protein fractions (106-18 kDa proteins) of B. abortus S2308 that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Twelve of the same 22 fractions, which contained 49-27 kDa proteins, produced a stimulation index of greater than 10 when incubated with LN cells taken from S19-vaccinated or SRB51-vaccinated cattle. Two factions, which contained 27 kDa proteins of S2308, induced the highest proliferative response (stimulation index 25 or greater) by LN cells in cattle given either S19 or SRB51. These results suggest that cattle vaccinated with S19 or SRB51 have similar LN immune responses to S2308, but unlike S19, SRB51 does not induce positive results in the standard tube agglutination test used to diagnose brucellosis in cattle.

Cloning bovine cytokine cDNA fragments and measuring bovine cytokine mRNA using the reverse transcription-polymerase chain reaction.

Bovine cytokine-specific primers and the reverse transcription-polymerase chain reaction (RT-PCR) were used to clone cDNA fragments that were specific for bovine IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma. Specificity of the cDNA fragments was verified by sequence analysis based on known bovine IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma gene sequences. In addition, RT-PCR was used to monitor cytokine mRNA expression in concanavalin A (Con A) and lipopolysaccharide (LPS)-stimulated bovine peripheral blood mononuclear cells (PBMC), and the results were compared with those obtained by measuring PBMC cytokine secretion using biologic assays. IL-1 activity in LPS-stimulated PBMC cultures was similar at 12 h and 24 h, although the activity decreased by approximately 40% at 48 h. IL-2 and IFN-gamma activity in supernatants of Con A-stimulated PBMC cultures was low at 12 h and reached maximum levels at 48 h. RT-PCR transcript analysis detected an increase in IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma mRNA expression that was usually correlated with the detection of these soluble cytokines by the bioassays. These results indicate that RT-PCR is a sensitive and effective method of obtaining cDNA probes and that this technique can be used to monitor bovine cytokine mRNA expression.

Experimental use of a dot-blot assay to measure serologic responses of cattle vaccinated with Brucella abortus strain RB51.

Brucella abortus strain RB51 was recently approved as an official brucellosis calfhood vaccine for cattle by the Animal and Plant Health Inspection Service branch of the United States Department of Agriculture. Currently available serologic surveillance tests for B. abortus do not detect seroconversion following SRB51 vaccination. The purpose of this study was to evaluate a dot-blot assay using gamma-irradiated strain RB51 bacteria for its specificity and sensitivity to detect antibody responses of cattle vaccinated with strain RB51. Dot-blot titers of sera at a recommended dosage (10(10) colony-forming units) were similar to those of sera from cattle vaccinated with similar numbers of B. abortus strain 19 and greater (P < 0.05) than titers of nonvaccinated cattle. In the first 12 weeks after vaccination with 10(10) colony-forming units of strain RB51, the RB51 dot-blot assay had 100% specificity for titers of 80 or less and a 53% sensitivity for titers of 160 or greater. Sensitivity of the RB51 dot-blot assay peaked at 4 weeks after vaccination with 10(10) colony-forming units of strain RB51. Dot-blot responses of sera from cattle vaccinated with a reduced dosage of strain RB51 (10(9) colony-forming units) did not differ (P > 0.05) from titers of sera from nonvaccinated cattle. Following intraconjunctival challenge with B. abortus strain 2308, titers on the RB51 dot-blot assay did not differ (P > 0.05) between nonvaccinated cattle and cattle vaccinated at calfhood with strain 19 or strain RB51.

Serologic responses of Brucella abortus strain 19 calfhood-vaccinated cattle following adult vaccination with strain RB51.

This study was designed to determine if Brucella abortus strain RB51, which expresses small amounts of the lipopolysaccharide O side chain, would cause positive responses on brucellosis serologic surveillance tests when given to adult cattle that were vaccinated as calves with B. abortus strain 19. Cattle vaccinated as adults with strain RB51 that had been vaccinated as calves with strain 19 (n = 40) had significantly greater antibody titers (P < 0.05) against strain RB51 at 4 and 8 weeks postvaccination in the dot blot assay than did animals (n = 10) not vaccinated with strain RB51. When evaluated using the card or buffered acid plate agglutination presumptive tests, 7 strain RB51 vaccinates tested positive at either 4 or 8 weeks following vaccination as compared with 4 cattle in the control group that were not vaccinated with strain RB51. One strain RB51 vaccinate was scored as suspect on the standard tube agglutination (STA) test at 8 weeks following vaccination. Remaining samples from strain RB51 vaccinates tested negative on the STA, complement fixation (CF), rivanol, and particle concentration fluorescence immunoassay (PCFIA) confirmatory tests. Samples from 2 control cattle were PCFIA positive at time 0; 1 of these animals was CF positive throughout the study. This study suggests that use of strain RB51 in cattle vaccinated with strain 19 as calves will not cause positive responses on confirmatory tests and will not impair brucellosis serologic surveillance efforts.

In vitro lymphocyte proliferative responses and gamma-interferon production as measures of cell-mediated immunity of cattle exposed to Pasteurella haemolytica.

Cell-mediated immune mechanisms may play a role in the pathogenesis and prevention of pneumonia in cattle caused by Pasteurella haemolytica serotype A1. To determine the circumstances required to stimulate and identify cell-mediated immune responses, calves were vaccinated with a commercial P. haemolytica bacterin or a live commercial P. haemolytica vaccine, or were infected intratracheally with virulent P. haemolytica. All calves were challenge-exposed intratracheally with P. haemolytica 31 d after vaccination or prior infection. Peripheral blood mononuclear cells and mediastinal and superficial cervical lymph node cells were stimulated with antigens prepared from P. haemolytica to evaluate in vitro proliferative responses and gamma-interferon production as measures of cell-mediated immunity. Strong proliferative responses and gamma-interferon production were detected in lymph node cells from calves vaccinated with the live vaccine and from infected calves, especially in response to stimulation with an outer membrane protein preparation from P. haemolytica. Greater proliferative responses and gamma-interferon production were associated with the lymph node nearer the site of bacterin administration (superficial cervical lymph node) or the site of infection (mediastinal lymph node), whereas greater proliferative responses and gamma-interferon production were associated with the more distant lymph node (mediastinal lymph node) in calves vaccinated with the live vaccine. Neither proliferative responses nor gamma-interferon production were detected in peripheral blood mononuclear cells from calves that were vaccinated for or infected with P. haemolytica. Antileukotoxin antibody titers were determined by a serum neutralization assay, and protection against pneumonic lesions was more closely correlated with antileukotoxin antibody responses than with lymphocyte proliferation or gamma-interferon responses.

Cytokine production during endotoxin-induced mastitis in lactating dairy cows.

The role of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha during endotoxin-induced mastitis in cows was characterized. Six cows had 10 micrograms of Escherichia coli lipopolysaccharide infused into 1 mammary gland. Three other cows served as nontreated controls. Within 1.5 to 2.5 hours after infusion, endotoxin caused obvious edema of the mammary gland and increased serum albumin concentration in milk of infused glands 6 times. Milk somatic cell count began to increase 3 to 5 hours after infusion in all treated glands. At 7 hours after infusion, somatic cell counts were increased > 10 times, compared with counts in milk from control cows. Pyrexia of > 1 C developed in only 1 cow, but all treated cows had serum cortisol concentrations > 50 ng/ml in response to endotoxin treatment. High concentrations of IL-1 (10 to 600 U/ml) and IL-6 (2 to 22 U/ml) were detected in milk of infused glands beginning 2.5 to 4 hours after infusion. Endotoxin did not induce detectable amounts of tumor necrosis factor activity in milk or serum. Swelling and mammary gland permeability changes preceded any detectable increase in IL-1 and IL-6 activity, indicating that these clinical signs of inflammation were not mediated by these cytokines. Systemic responses and the leukocytic influx into endotoxin-infused glands developed after or concurrently with initial increases in IL-1 and IL-6 activities in milk. These results suggested that IL-1 and IL-6 may have a role in mammary gland defenses and in the pathophysiologic changes during endotoxin-induced mastitis.

Monophosphoryl lipid A-induced immune enhancement of Brucella abortus salt-extractable protein and lipopolysaccharide vaccines in BALB/c mice.

A study was conducted to determine the effect of monophosphoryl lipid A (MPL) and trehalose dimycolate (TDM) as adjuvants on the protective responses in BALB/c mice vaccinated with Brucella abortus salt-extractable protein (BCSP) or proteinase-K-treated B abortus lipopolysaccharide (PKLPS). Mice were vaccinated with different doses of BCSP or PKLPS given alone or in combination with MPL or TDM. Mice were challenge-exposed 4 weeks later with virulent B abortus strain 2308. Two weeks after challenge exposure, the number of B abortus colony-forming units (CFU) per spleen, spleen weights, and spleen cell interleukin 1 production were measured. Serum IgG and IgM concentrations specific for vaccinal immunogens were measured before and after challenge exposure with B abortus. Spleen weights and mean B abortus CFU per vaccine group were significantly lower in BCSP- and PKLPS-vaccinated mice, compared with those of nonvaccinated control mice. Monophosphoryl lipid A enhanced the suppression of splenic infection when given with the BCSP vaccine, but not when given with the PKLPS vaccine. Trehalose dimycolate had no effect on mean CFU when given with BCSP, but incorporation of TDM resulted in a significant increase in mean CFU when given with PKLPS. Spleen weights in BCSP- or PKLPS-vaccinated mice were not different when these vaccines were combined with MPL or TDM. Because of the wide variation in the results, we could not conclude that vaccination with BCSP or PKLPS alone, or in combination with MPL altered spleen cell interleukin-1 production in B abortus-infected mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of serologic responses, lymphocyte proliferative responses, and clearance from lymphatic organs after vaccination of bison with Brucella abortus strain RB51.

OBJECTIVE: To evaluate clearance of the vaccine strain, immunologic responses, and potential shedding of Brucella abortus strain RB51 organisms after vaccination of bison calves. ANIMALS: Fourteen 7-month-old female bison calves. PROCEDURE: 10 bison calves were vaccinated SC with 1.22 x 10(10) colony-forming units of B abortus strain RB51. Four bison calves were vaccinated SC with 0.15M NaCl solution. Rectal, vaginal, nasal, and ocular swab specimens were obtained to evaluate potential shedding by vaccinated bison. The superficial cervical lymph node was biopsied to evaluate clearance of the vaccine strain. Lymphocyte proliferative responses to strain RB51 bacteria were evaluated in lymph node cells obtained from biopsy specimens and also in peripheral blood mononuclear cells. RESULTS: Strain RB51 was recovered from superficial cervical lymph nodes of vaccinates examined 6, 12, and 18 weeks after vaccination (4/4, 3/4, and 1/4, respectively) but not in vaccinates examined at 24 weeks (0/3) after vaccination or nonvaccinates examined at all sample collection times (n = 1 bison/sample period). Serologic, immunologic, and bacterial culture techniques failed to reveal shedding of strain RB51 by vaccinates or infection of nonvaccinated bison. Lymphocyte proliferative responses were evident in lymph node cells and blood mononuclear cells from strain RB51-vaccinated bison beginning 12 weeks after vaccination. CONCLUSION: Strain RB51 was cleared from bison by 18 to 24 weeks after vaccination. Bison vaccinated with strain RB51 did not shed the vaccine strain to nonvaccinated bison housed in close proximity. Strain RB51 did not induce antibody responses in bison that would interfere with brucellosis surveillance tests, but did stimulate cell-mediated immunity.

Immune responses and protection against infection and abortion in cattle experimentally vaccinated with mutant strains of Brucella abortus.

Twenty-four 10-month-old Polled Hereford heifers were inoculated SC with live cells of one of the following strains of Brucella abortus: S19 delta 31K (n = 4), S19 delta SOD (n = 4), RB51 (n = 4), and strain 19 (n = 6); controls (n = 6) were given saline solution. Heifers given the deletion mutants S19 delta 31K and S19 delta SOD, and those given strain 19 developed antibody responses to B abortus and cutaneous reactions to brucellin. Heifers given strain RB51 did not develop antibodies that reacted in the standard tube agglutination test, but sera reacted in tests, using an antibody dot-blot assay containing RB51 antigen. The S19 delta 31K and S19 delta SOD strains of B abortus isolated from lymph node tissue after vaccination did not differ genetically from the master stock strain. All heifers were bred naturally at 16 to 17 months of age, and were challenge-exposed intraconjunctivally with virulent B abortus strain 2308 during the fifth month of pregnancy. All vaccinated heifers were protected (ie, none aborted and none had B abortus isolated from their tissues after parturition). Calves born from vaccinated dams were free of B abortus. Antibody responses in heifers after challenge exposure were an indicator of immunity. All 5 control heifers (nonvaccinated) developed serum antibodies after challenge exposure; 3 aborted, and 1 delivered a small, weak calf at 8.5 months of gestation. Thus live mutant strains of B abortus can induce protective immunity when given at 10 months of age, and strain RB51 is a strong candidate for further testing.