Vaccination with gamma-irradiated Neospora caninum tachyzoites protects mice against acute challenge with N. caninum.

Neospora caninum, an apicomplexan parasite, is a leading cause of bovine abortions worldwide. The efficacy of gamma-irradiated N. caninum strain NC-1 tachyzoites as a vaccine for neosporosis was assessed in C57BL6 mice. A dose of 528 Gy of gamma irradiation was sufficient to arrest replication but not host cell penetration by tachyzoites. Female C57BL6 mice were vaccinated with two intraperitoneal inoculations of 1 x 10(6) irradiated tachyzoites at 4-wk intervals. When stimulated with N. caninum tachyzoite lysates, splenocytes of vaccinated mice, cultured 5 and 10 wk after vaccination, secreted significant (P<0.05) levels of interferon gamma, interleukin (IL)-10, and small amounts of IL-4. Antibody isotype-specific ELISA of sera from vaccinated mice exhibited both IgG1 and IgG2a isotypes of antibodies. Vaccinated mice were challenged intraperitoneally with 2 x 10(7)N. caninum tachyzoites. All vaccinated mice remained healthy and showed no obvious signs of neosporosis up to the 25th day post-challenge when the study was terminated. All unvaccinated control mice died within 1 wk of infection. Gamma-irradiated N. caninum tachyzoites can serve as an effective, attenuated vaccine for N. caninum.

Frequent exposure of mice to crude Brucella abortus proteins down-regulates immune response.

Mice repeatedly immunized via the intraperitoneal route with a Brucella abortus antigen lost their ability to develop a strong in vitro lymphoproliferative response. This result correlates with a decreased tendency of the lymphoid population to produce interferon-gamma when stimulated in culture with the immunizing antigen. With respect to the humoral response, as the number of immunizations increased, the animals produced more specific immunoglobulin M and immunoglobulin G1 antibodies. It is postulated that the long-term exposure of an animal to Brucella antigen changes the nature of the immune response from a T-cell-mediated response to a humoral response favouring the establishment of the disease.

Effect of copper and iron on neutrophil function and humoral immunity of gestating beef cattle.

Eighty gestating beef cattle were used to determine the effect of trace mineral salt mixtures containing copper (Cu) and iron (Fe) on selected immune functions and factors affecting copper bioavailability. Pastured cattle were randomly assigned to receive one of the following combinations of Cu and Fe in the free-choice trace mineral salt: (1) 0 mg of Cu/0 mg of Fe/kg of trace mineral salt, (2) 1,600 mg of Cu (CuSO4)/3,000 mg of Fe/kg of trace mineral salt, (3) 1,600 mg of Cu (CuSO4)/0 mg of Fe/kg of trace mineral salt, and (4) 1,600 mg of Cu (CuCO3)/3,000 mg of Fe/kg of trace mineral salt. Total Cu/Fe consumption (from trace mineral salt) was 2/678, 193/1,050, 162/553, and 202/1,140 mg/head/d, respectively, for the 4 groups. After a 1-month period of acclimation and also on day 28 of the 36-day study, copper concentrations in serum were significantly (P < 0.05) lower in group 1 than in groups 3 and 4. Serum copper concentrations did not increase with time for any group, whereas hepatic copper concentrations increased significantly (P < 0.05) with time for all groups except group 1. Hepatic iron concentrations were similar among groups at the time of the initial and final hepatic biopsies on days 0 and 28, respectively. Hepatic iron concentrations increased significantly (P < 0.05) with time in groups 3 and 4. Humoral response to chicken gamma-globulin was high but did not differ among groups on any of the days analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial survival, lymph node changes, and immunologic responses of cattle vaccinated with standard and mutant strains of Brucella abortus.

Forty-eight cattle were used in 4 experiments; 6-week-old calves in experiments 1-3 (n = 24) and 10-month-old heifers in experiment 4 (n = 24). In experiments 1-3, 7 groups of 3 calves each were inoculated SC with 5 strains of Brucella abortus: virulent strain 2308 (2 groups), vaccine strain 19 (2 groups), and mutant strains RB51. 19 delta 31K, and 19 delta SOD. Sera and lymph node tissues were examined at 2-week intervals for evidence of infection. At postinoculation (PI) week 12, 2 calves in each group were given dexamethasone for 5 days. Calves were then euthanatized and lymphoid tissue, spleen, liver, and bone marrow were examined for evidence of B abortus. Calves given strain 2308 had large numbers of bacteria in their lymph nodes, marked granulomatous lymphadenitis in the deep cortex, and loss of lymphoid cells in superficial cortical areas. In addition, they had high serum antibody titers at PI week 16. Calves given strain 19, or genetic mutants derived from strain 19, cleared bacteria from lymph nodes more rapidly, had less lymphoid destruction, and developed antibody titers that did not persist for 16 weeks. The RB51 strain (rough) was cleared most rapidly from lymphoid tissues and induced serum antibody responses only to the core of the lipopolysaccharide molecule. Treatment of calves with dexamethasone did not cause B abortus to reappear in tissues of any calves, nor did serum antibody titers increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of monoclonal antibodies directed against swine leukocytes.

Hybridomas were produced from fusions of the SP2/0 mouse myeloma with splenic cells from: 1) an outbred Sprague Dawley rat immunized with swine peripheral blood mononuclear (PBM) cells; 2) a (CBA/NDub X BALB/c Dub) F1 mouse immunized with concanavalin A (Con A) activated swine PBM cells and 3) a (BALB/c Dub X C3H/He Dub) F1 mouse immunized with swine thymocytes. The resulting supernatants were screened by a microcytotoxicity assay for activity against swine PBM cells. Four hybridomas (MSA1, MSA2, MSA3 and MSA4) were selected, cloned and characterized by their cell reactivity and effect on mitogenic assays. MSA1 and MSA2 belong to the rat IgG2b subclass. MSA3 and MSA4 are of the mouse IgG2a subclass. These monoclonal antibodies reacted in the following manner: MSA1 with monocytes, granulocytes, red blood cells and bone marrow cells; MSA2 with subset of T cells; MSA3 with B cells and subsets of T cells and monocytes (class II molecule) and MSA4, a pan-T cell reagent (E-rosette receptor). The involvement of the various cell types reactive to the different monoclonal antibodies in the mitogenic response of swine PBM cells to Con A, phytohemagglutinin (PHA) or pokeweed mitogen (PWM) was investigated by cellular depletion with monoclonal antibody plus complement. Cellular depletion of PBM cells with the following monoclonal antibodies plus complement treatment resulted in: MSA1, almost total reduction in the mitogenic response to low doses of Con A or PWM; MSA2, partial reduction in the proliferative responses to any concentration of Con A, PHA or PWM; MSA3, partial reduction in proliferative responses to low concentrations of Con A or PWM and 4) MSA4, total elimination of any proliferative response to Con A, PHA or PWM.

Monoclonal antibodies to Brucella surface antigens associated with the smooth lipopolysaccharide complex.

Hybridomas producing antibodies to determinants associated with the lipopolysaccharide (LPS) of Brucella abortus and B melitensis were obtained by polyethylene glycol fusion of the SP2/0 myeloma cell line with B lymphocytes harvested from a Sprague-Dawley-derived rat previously immunized with whole B abortus strain 1119 organisms. Two clones, BRU38 and BRU28 , were selected for their ability to react with whole B abortus organisms and purified smooth-LPS ( f5p ). The BRU38 monoclonal antibodies were absorbed with live, rough strain 45/20 and smooth strains of B abortus and B melitensis organisms, whereas only smooth strains absorbed the antibody activity from BRU28 . Complete inhibition of the monoclonal's activity could be achieved with crude smooth-LPS, a purified f5p fraction, and a water-soluble acid degraded polysaccharide. Absorption of BRU38 and BRU28 with rough Brucella LPS, polysaccharide-B antigen, keto- deoxyoctanoic acid, or with several sugars and fatty acids known to be components of the Brucella LPS complex had no effect on the monoclonals. The data indicate that antigenic determinants are associated with the smooth LPS complex, probably with the O-side chain, and are expressed patchwise and in different quantities on several strains of B abortus and B melitensis. The B abortus rough strain 45/20 contains surface determinants which lead to the agglutination of smooth strain 1119 organisms. The potential use of monoclonals in competitive enzyme-linked immunosorbent assays for diagnostic purposes is discussed.

Heterogeneity of contagious ecthyma virus isolates.

Results of cross-neutralization tests of 4 isolates of contagious ecthyma (CE) virus and their antisera indicated that the isolates were neutralized to various degrees by the CE virus antisera. Cross-reactions among isolates were unilateral, but not bilateral, and therefore grouping of CE virus isolates by neutralization tests was not possible to obtain. Structural analysis of polypeptides from 11 isolates of CE virus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the profiles obtained were similar to one another, except for differences observed in the molecular weight region of 37,000 to 44,000. On the basis of these differences, the isolates could be classified into 4 groups. The polypeptides which varied among the different isolates were shown to be in the surface component of the virion and appeared to be components of the surface tubules. Unilateral cross-reactions detected in cross-neutralization tests were found to correlate with the 37,000 to 44,000 dalton polypeptide grouping of the isolates.

Genotypic, phenotypic, and biological characteristics of Moraxella bovis.

Several isolates of Moraxella bovis obtained from cattle with infectious bovine keratoconjunctivitis killed monocytes and macrophages in vitro and carried 3 to 5 plasmids. Cloned and noncloned M bovis isolates readily induced the disease, killed the phagocytes in vitro, and carried 3 plasmids. Moraxella bovis isolates of low in vivo virulence carried 5 plasmids and did not kill phagocytes to the extent that isolates with 3 plasmids did. The possible implications of these findings in the pathogenesis of infectious bovine keratoconjunctivitis are discussed. Also, a classification of M bovis colonies into rough or smooth varieties according to their staining characteristics with crystal violet is suggested.

Hemorrhagic enteritis in turkeys: purification and quantification of the virus.

Two methods for purifying the virus of hemorrhagic enteritis from infected turkey spleens are described. One procedure utilized precipitation with polyethylene glycol, and the other consisted of trichlorotrifluoroethane extraction. Both procedures included sucrose-cesium chloride gradient centrifugation in the final purification step. The buoyant density of the viral fraction was 1.34 g/cm3, typical for adenoviral particles, and the size and morphologic characteristics of the virions observed by transmission electron microscopy suggested that the purified virus belongs to the family Adenoviridae. The biologic activity of the purified virus was titrated by inoculating 10-fold dilutions of the viral suspension into turkey poults. Mortality and hemorrhagic diarrhea proved to be inconsistent parameters of infection, and the degree of splenomegaly was proportional to the virus dose. The body/spleen ratio was the parameter selected for measuring viral activity, and the body/spleen ratio 50% was adopted as the unit for the titration of the virus. By using the same system it was demonstrated that the infectivity of the virus could be neutralized with antiserum produced in turkeys.

Enzyme-linked immunosorbent assay for measurement of anti-Moraxella bovis antibodies.

Antibodies of the immunoglobulin (Ig) G and IgA classes specific for Moraxella bovis in serum or lacrimal secretions of cattle were detected with an enzyme-linked immunosorbent assay, using soluble extracts of M bovis or whole organisms. The amount of detectable antibodies was influenced by the type of antigenic preparation used in the enzyme-linked immunosorbent assay. The test indicated that 2 subcutaneous inoculations of M bovis cells induce significantly (P less than 0.0001) increased anti-M bovis serum antibodies. The infection of calves with M bovis induced specific IgG antibodies in serum and predominantly IgA antibodies in lacrimal secretions.

Enzyme-linked immunosorbent assay for bovine immunoglobulin subclass-specific response to Brucella abortus lipopolysaccharides.

An enzyme-linked immunosorbent assay was developed to follow the bovine response, by immunoglobulin class and subclass, to defined smooth and rough lipopolysaccharides (LPS) of Brucella abortus. Binding to smooth LPS of immunoglobulin G1 (IgG1) and IgG2 in sera from Brucella-infected animals was significantly greater than binding in sera from normal uninfected animals. Competition or steric blocking among IgM, IgG1, and IgG2 for binding sites on smooth LPS was shown to occur. Binding of IgM to Brucella smooth LPS with sera from uninfected animals was elevated above the assay control levels, and attempts to eliminate this nonspecific IgM binding were not successful. The same levels of nonspecific IgM binding were also seen with Brucella rough LPS, Escherichia coli LPS, and Pseudomonas solanacearum LPS. Sera from some, but not all, Brucella-infected animals showed elevated binding of IgG1 and IgM to both E. coli LPS and Brucella rough LPS as well as to Brucella smooth LPS. This was interpreted as specific antibody. Cross-reactions between B. abortus smooth or rough LPS and E. coli LPS could not be shown by immunodiffusion.

Bovine venereal vibriosis. Ultrastructure of endometrial inflammatory lesions.

The inflammatory lesions in bovine venereal vibriosis have been examined by light and transmission electron microscopy in order to provide a better understanding of host defense of the uterus against bacterial invasion. Neutrophils and eosinophils were found mainly at the endometrial surface and in glandular lumina. Mononuclear cells within the endometrial tissue were identified ultrastructurally. Lymphocytes were most abundant, plasma cells were next in frequency, and mononuclear phagocytes were least often observed. An attempt was made to classify lymphocytes on the basis of presence or absence of a cytoplasmic network of filaments and other ultrastructural characteristics.

Immunoglobulin classes and biological functions of Campylobacter (Vibrio) fetus antibodies in serum and cervicovaginal mucus.

Serum and cervicovaginal mucus (CVM) antibodies from heifers after genital infection or systemic immunization with Campylobacter (Vibrio) fetus were classified according to their immunoglobulin class, antigenic specificities, and biological functions. Only immunoglobulin (Ig) A antibodies, specific both for O and superficial, heat-labile, whole-cell (W) antigens, were detected in CVM of convalescent animals. After systemic immunization, antibodies in serum were directed principally to W antigens and were located in IgG(1), IgG(2), and IgM classes; CVM antibodies of the same specificity were detected only in the IgG subclasses. Functional tests revealed that antibodies of W specificity, whether of the IgA or IgG class, were capable of immobilizing the organism. However, IgG antibodies immobilized with clumping, whereas IgA antibodies immobilized single organisms within the 3-min period. None of the antibody preparations was bactericidal in the presence of homologous complement when the infecting strain was used as the target organism, but a bactericidal effect was observed when the target strain was rough and non-encapsulated. Both serum and CVM from systemically immunized animals opsonized C. fetus organisms, but CVM from locally immunized animals containing IgA antibodies was not opsonic. It is hypothesized that functions of immobilization for IgA and IgG and of opsonization for IgG are important features of protective immunity in venereal vibriosis.