Genus-, subspecies-, and serotype 1-specific antigens of Chlamydia psittaci were characterized by immunoblot analysis, using monoclonal antibodies that recognize 2 C psittaci strains: AB7 isolated from an ewe that had aborted, and iB1 isolated from feces of a healthy ewe. Genus-specific epitopes were detected on lipopolysaccharide, on a 47-kd protein, and on a 27- to 30-kd doublet. Subspecies-specific epitopes were located on a 30-kd protein, and a 80- to 90-kd protein region was identified, which bore subspecies- and serotype 1-specific epitopes. These 80- to 90-kd proteins were highly reactive with serum from ewes that had aborted and could be a useful antigen for diagnosis of chlamydial induced abortion of ruminants.

Polypeptides from whole cell preparations of Pasteurella multocida serotypes A:12 and A:3 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Antigens were detected by immunoblot analysis, using sera from 3 groups of rabbits. Sera were obtained from rabbits inoculated intranasally with P multocida serotype A:12 or A:3, from rabbits maintained in a rabbitry with enzootic P multocida A:12 infection, and from rabbits maintained in a rabbitry with enzootic P multocida A:3 infection. Immunoblot analyses of pre- and postinoculation sera from experimentally infected rabbits, using serotype A:12 antigen, revealed 3 polypeptides with approximate molecular mass of 28, 30, and 37 kDa that consistently detected antibodies after P multocida-induced infection. Sera from rabbits naturally infected with either serotype, tested against serotype A:12 and A:3 antigens, detected the same polypeptides in both serotypes. Thus, immunologic reactivity to these polypeptides may be useful for serologic detection of P multocida infection.

In an attempt to establish if cross protection can be induced by different strains of Haemophilus parasuis, three groups of 12 gnotobiotic pigs were immunized each with an aluminum hydroxide adsorbed whole cell bacterin of one of three H. parasuis strains. Two weeks later, four pigs within each vaccinated group were challenged with aerosols of live cultures of each of the three test strains and observed for response. Two virulent strains V1 and V2 protected all the vaccinated pigs, while all non-vaccinated controls succumbed to Glasser's disease when challenged with these strains. Vaccination with strain LV (of low virulence) protected the pigs against challenge with strain V2, but not against strain V1. Strain LV did not cause disease in the immunized animals and only in one of ten nonimmunized pigs upon second challenge. The results suggest that strains may differ in antigenicity and that virulence and immunoprotection are positively related. Strains to be used in commercial vaccines should therefore be selected carefully. Antibodies detected in the sera of vaccinated pigs were to outer membrane proteins of the bacteria, but not to lipopolysaccharides or capsular polysaccharides. This would suggest that for gnotobiotic pigs outer membrane proteins are more immunogenic than lipopolysaccharide or capsular antigens. Further work is needed to determine if outer membrane proteins also contribute protective immunogens.

Thirty-seven local isolates of Actinobacillus suis-like organisms from diseased and clinically normal horses and 1 Ilama were compared with reference strains of A suis, A lignieresii, A equuli, A capsulatus, A hominis, A (Pasteurella) ureae, and equine A suis-like organisms (ASLO) previously described in literature. Comparison was by cultural characteristics, carbohydrate fermentation, enzyme profiles, and whole-cell protein polyacrylamide gel electrophoresis. Carbohydrate fermentation, determined by API-CH gallery, divided 36 equine ASLO isolates into 6 API-CH biotypes. The Ilama isolate was an additional distinct biotype. The biochemical comparisons between A suis and ASLO did not reveal remarkable and consistent differences. Enzyme analysis revealed 5 API-ZYM biotypes, one of which included the same strains as one of the API-CH biotypes and consisted in both instances of 4 esculin-negative ASLO cultures and the reference strain of A lignieresii. We conclude that the 4 strains were hemolytic variants of A lignieresii. Protein electrophoresis disclosed 15 banding patterns, 10 of which represented equine ASLO strains. The reference strains of A suis shared the pattern predominant among equine ASLO. Four of the remaining reference strains of Actinobacillus species each had a unique profile, whereas the type strain of A capsulatus and the Ilama isolate had similar profiles. The groupings of cultures resulting from the different testing methods had little relation to each other and to the anatomic source of the strains except the strains comprising API-CH biotype III, which originated in the equine respiratory tract, and the A lignieressi cluster.

A double-blind randomized clinical trial was undertaken to determine the value of parenterally administered Streptococcus equi M-protein vaccine in foals during an epizootic of strangles. Weaned mixed-breed foals (n = 664) housed on 2 adjacent feed-lots (A and B) arrived over a 5-day period, 2 weeks before primary vaccination. Foals in lot B (n = 114) were randomly administered vaccine (n = 59) or saline solution (placebo; n = 55) on 3 occasions at biweekly intervals. Foals in lot A (n = 450) were given 1 dose of vaccine (n = 225) or placebo. The following clinical observations were scored blindly by a single observer for all foals in lot B and for 120 (randomly sampled) foals in lot A on a single day, 2 (lot B) and 6 (lot A) weeks after final vaccination: cervical lymphadenopathy, type of bilateral nasal discharge, and palpable swelling at injection site(s). Bacteriologic culture of nasal swab specimens or lymph node aspirates from selected foals with clinical disease yielded S equi. Cervical lymphadenopathy was observed in 17 of 59 (29%) vaccinates and 39 of 55 (71%) nonvaccinated controls in lot B and in 32 of 60 (53%) vaccinates and 29 of 60 (48%) controls in lot A. Contingency X2 analysis confirmed significantly lower cervical lymphadenopathy rate (X2 = 18.5; P < 0.001) and prevalence of mucopurulent nasal discharge (X2 = 11.4; P < 0.01) for vaccinates in lot B only. Swelling(s) at the vaccine injection site were palpated in 44% of lot B and 29% of lot A vaccinates vs < 2% of placebo controls. In the face of intense natural exposure, foals inoculated 3 times with M-protein vaccine were less than half as likely to have clinical signs of strangles as were nonvaccinated horses.

A study was conducted to determine whether subcomponent proteins (previously identified as BCSP20, BCSP3l, and BCSP45, and the corresponding recombinant proteins rBCSP20, rBCSP31, and rBCSP45) that were recovered from the cell surface of Brucella abortus strain 19 were immunogenic and protective for mice when compared with Brucella cell surface protein (BCSP) and with a proteinase K-treated lipopolysaccharide (PKLPS) extracted from B abortus strain 2308. Protection was evaluated after challenge exposure with a virulent culture of B abortus strain 2308, using CD-1 or BALB/c mice or both inoculated with vaccines of various combinations and concentrations, with and without PKLPS or BCSP. Protection was assessed by enumeration of splenic colony-forming units, reduced mean splenic weight relative to controls, and the relative serologic responses (immune response) in an ELISA. The general results indicate that BCSP, PKLPS, BCSP20, and BCSP31 are immunogenic or protective or both. Protectiveness was not observed for each of the recombinant proteins; however, results from the combined recombinant protein vaccine study suggest the immunogenicity of the recombinant proteins. The apparent immune-inducing properties of BCSP20 and BCSP3l are thought to be attributable to the presence of an immunogenic and protective BCSP fraction (possibly lipopolysaccharide) still associated. Serologic results support our conclusion that each of the recombinant protein vaccines did not induce a protective response comparable to that of BCSP or PKLPS, even when the subcomponents were combined. Although the results suggest that the subcomponents of BCSP apparently induced partial protection, they are thought to be only a part of the antigens contained in BCSP that influence the serologic response. Our findings may serve as an experimental model to determine the mechanisms involved in the protective responses induced by Brucella antigens.

Alterations in the size and functions of porcine alveolar macrophages exposed to sublytic amounts of heat-labile, hemolytic cytotoxin produced by Actinobacillus pleuropneumoniae (App) serotype 1, strain HS54 into the culture medium were studied in vitro. Alveolar macrophages were sensitive to the cytotoxin; treatment of the macrophages with low concentrations of cytotoxin (0.016 hemolytic unit) resulted in severe, irreversible cell swelling. However, high doses of cytotoxin (2.0 hemolytic units) were required to cause substantial cell death, as indicated by the influx of propidium iodide into and release of lactate dehydrogenase from cells. Macrophages exposed to low, sublytic doses of cytotoxin failed to migrate toward chemoattractant, were unable to attach to glass, and failed to phagocytize optimally opsonized erythrocytes. Macrophages already attached to glass surfaces detached when exposed to sublytic doses of cytotoxin. The swelling and impairment of functions of alveolar macrophages observed in this study could not be attributed to endotoxic effects, because heat treatment of the cytotoxin preparation for 60 minutes at 60 C resulted in complete loss of cytotoxicity. We conclude that sublytic doses of heat-labile, hemolytic cytotoxic substances produced by App depress alveolar macrophage function at concentrations likely to develop in association with acute pulmonary infection with App. The Apx (A pleuropneumoniae Rtx toxins) exotoxins secreted by the bacteria into culture medium were considered responsible for the toxic activity of the cytotoxin preparation. The Apx of the App field strain used in this study were likely to be similar to those of serotype-1 reference strain (S4707). Analysis by use of DNA-DNA hybridization indicated that genomic DNA of the field strain contained sequences similar to those encoding structural protein of ApxI (apxIA) and ApxII (apxIIA) of the serotype-1 reference strain. Therefore, Apx produced by the field strain of App used in this study are likely to be of similar pathogenic importance worldwide.

A challenge-exposure model was developed for dose-dependent induction of subclinical (moderate) atrophic rhinitis (AR) in conventionally raised Dutch Landrace and Large White pigs, about 4 weeks old. Under favorable climatic and housing conditions, pigs were intranasally challenge-exposed with Pasteurella multocida-derived toxin (Pm-T) 3 days after pretreatment by inoculation with 1% acetic acid. Pigs were challenge-exposed with 1 of the following Pm-T doses: 0 (control), 5, 13, 20, or 40 microgram of Pm-T/ml of phosphate-buffered saline solution (PBSS), 0.5 ml/ nostril/d on 3 consecutive days. Five weeks after challenge exposure, subclinical moderate) AR status was defined as intermediate conchal atrophy (grade 2 for ventral conchae on a 0 to 4 scale and grade 1 or 2 for dorsal conchae on a 0 to 3 scale, respectively) and perceptible difference in change in brachygnathia superior (CBS) between control and challenge-exposed pigs between the beginning and end of the study. All Pm-T-exposed pigs had nasal damage that was dose-dependent. The higher Pm-T doses resulted in higher ventral conchae atrophy and dorsal conchae atrophy scores. The CBS increased with applied Pm-T dose, resulting in significant (P < 0.05) differences between controls (3.88 mm) and the 13-, 20-, and 40-microgram Pm-T-treated groups (7.77, 6.58, and 7.98 mm, respectively). In response to the applied dose, weight gain per week for Pm-T-exposed pigs was lower than that of controls after week 3 (P < 0.01). Difference from controls was 32, 54, 52, and 96 g/d/pig for 5-, 13-, 20-, and 40-microgram Pm-T-treated groups respectively, in the last 2 weeks. For Dutch Landrace and Large White pigs, intranasally administered Pm-T mimicked the pathogenic effect of in vivo infection with toxigenic Pm strains. The optimal model to induce subclinical AR appeared to be 13 microgram of Pm-T/ml (0.5 ml/nostril/d) on 3 consecutive days. Our model should enable studies of exogenous and endogenous factors involved in development of AR, independent of the colonizing ability of the Pm strain used.

A study was conducted to determine the effect of mono-phosphoryl 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. Increased host protection as defined by decreased CFU could not be related consistently to increased BCSP- or PKLPS-specific serum IgG or IgM antibodies introduced by any of the vaccines. These results do not eliminate a role for antibodies in the protection observed.

Membrane associated proteins from 8 untypeable Pasteurella haemolytica strains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and compared with those of P haemolytica serotypes 1 and 2. Cattle antisera obtained from P haemolytica serotype 1 vaccine trials were used in immunoblotting assays to compare the membrane proteins from the 8 untypeable strains with those from P haemolytica serotypes 1 and 2. Densitometry was used to identify bands, and using linear regression analyses, the peak area optical densities (measuring antibody response) were correlated to lesion scores from the vaccinated calves. Significant antibody responses to proteins of 99, 69, 60, 55, 47, 45, 39, 33, 30, 16, and 14.5 kDa were detected for 4 or more of the 8 P haemolytica untypeable strains. Serotypes 1 and 2 of P haemolytica contained a comigrating 30-kDa protein. Antibody responses to proteins of 39, 33, and 32.5 kDa were significant for 3 of the untypeable strains and had significant correlation to lesion scores. Antibody responses to various other proteins were significant for 2 untypeable strains each.