Live, avirulent Escherichia coli vaccine strains were constructed and tested for efficacy in preventing colibacillosis in 4-week-old pigs. Either or both of 2 plasmids were inserted into avirulent E coli strain G58-1 (0101:NM). These plasmids were pPMC4, which encodes for LTb subunits of heat-labile enterotoxin, and pDHF1, which encodes for K88ac fimbriae. Litter- and weight-matched pigs were removed from sows when they were 10 days old and vaccinated orally with the constructed strains or with G58-1 (negative control vaccine) when they were 2 weeks old and 5 days later. All pigs were challenge-inoculated with virulent E coli strain 3030-2 (0157:K88, LT+, STb+) 2 weeks after the first vaccination. Only 1 pig vaccinated with G58-1/pPMC4/pDHF1 developed diarrhea and none died following challenge inoculation. Seventeen of 31 control pigs developed diarrhea and 11 died. Of 18 pigs vaccinated with G58-1/pDHF1 then challenge-inoculated with the virulent strain, 5 developed diarrhea and 2 died. Fifteen of 18 litter- and weight-matched controls developed diarrhea and 8 died. When compared with G58-1 (negative control), G58-1/pPMC4 afforded no protection to pigs challenge-inoculated with 3030-2.

Sublethal irradiation of BALB/c mice 4 hours prior to inoculation with 5 X 10(4) 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.

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.

Oral keratinocytes from dogs were cultured on either collagen gels or artificial matrices at the air-liquid interface, and the expression of keratinocyte antigens and basement membrane components was determined, using various monoclonal and polyclonal antibodies. Keratinocytes grown on collagen gels expressed pemphigus vulgaris, pemphigus foliaceous, and bullous pemphigoid antigens. Diffuse, suprabasal, and superficial keratinocyte membrane differentiation antigens identified by various monoclonal antibodies also were expressed in a pattern identical to that observed in the native tissue. Laminin and type-IV collagen were deposited at the keratinocyte-collagen interface in a patchy distribution. When synthetic matrices were used, the oral keratinocytes differentiated, but to a lesser extent than cells grown on collagen gels. Antigen expression for cells grown on synthetic matrices was similar to that for cells on collagen, except for failure of the keratinocytes on synthetic membranes to express superficial cell antigens and pemphigus foliaceous antigens.

Chemotaxis under agarose was evaluated to establish an assay system and to characterize chemotacticresponses of canine neutrophils. A method for the measurement of canine neutrophil chemotaxis was established, with optimal responses obtained with agarose containing 10% pooled canine serum, a concentration of 5 X 10(5) cells/well, zymosan-activated serum (ZAS), or autologous serum or plasma as the chemoattractants, and a 120-minute incubation period. Canine neutrophils responded well to ZAS, heat-inactivated ZAS, autologous serum and plasma, and heat-inactivated pooled serum. Chemotactic activity was proportional to the concentration of serum used as the chemoattractant. Mean (+/- SD) random migration, chemotaxis, chemotactic index, and chemotactic differential of neutrophils from 9 healthy Greyhounds were 1.09 (+/- 0.23), 1.95 (+/- 0.38), 1.82 (+/- 0.31), and 0.86 (+/- 0.32) mm,respectively.

The relative sensitivity of bovine blood monocytes and macrophages isolated from milk to lipopolysaccharide, with respect to interleukin 1 (IL-1) production, was evaluated. Addition of lipopolysaccharide (0 to 30 microgram/ml) to theculture medium resulted in increases in secreted and intra-cellular IL-1 activity for monocytes and milk macrophages, with maximal stimulation achieved at 30 microgram oflipopolysaccharide/ml of medium. At this concentration of lipopolysaccharide, monocytes released 76% of the total IL-1, whereas milk macrophages released only 26% of the total IL-1 produced within the cell. Secretion of a small quantity of IL-1 was a common property of macrophages isolated from healthy and mastitic quarters. We concluded that limited secretion of IL-1 may render the milk macrophages less efficient in promoting lymphocyte activation.

To assess the effect of ivermectin on immune function (antibody production), male CD-1 mice were inoculated with an antigen the day after SC administration of ivermectin (0.2 mg/kg of body weight or 20 mg/kg). Responses were evaluated 5 days after inoculation of the antigen. Antibody production against sheep RBC, a T lymphocyte-, macrophage-dependent response, was enhanced by ivermectin treatment (P = 0.00049). In contrast, antibody production against dinitrophenyl-Ficoll, a T lymphocyte-independent, macrophage-dependent response, was not altered by ivermectin treatment. Results indicate that the immunostimulatory properties of ivermectin are associated with altered function of T lymphocytes, in particular, T-helper lymphocytes. The immunomodulating effects of ivermectin may provide an alternative approach for treatment of disease problems involving immunosuppression.

Phagocytic and nitroblue tetrazolium (NBT) reductive activities of blood neutrophils from 19 Holstein heifers were measured by light microscopic and spectrophotometric methods, respectively. These functional properties of neutrophils correlated well (r = 0.64) and varied significantly (P < 0.05) among animals studied. Variations in phagocytosis and NBT reductive activities attributable to the source of sera were determined in experiments in which cells from the same cows and zymogen particles opsonized with heat-inactivated autologous or homologous sera were used. Variations attributable to the source of cells were determined in experiments in which cells from different cows and particles opsonized with pooled sera from all the cows were used. Most of the variation in phagocytic properties and NBT reductive activities was attributable to the source of cells (ie, each cow). The source of sera contributed slightly to the variation in NBT reductive activities, but not to the phagocytic properties. These results support the concept of functional heterogeneity of neutrophils among cows.

Two commercially available synthetic adjuvant systems, trehalose dimycolate (TDM) and TDM + monophosphoryl lipid A (MPL), were compared with Freund complete adjuvant (FCA) for the ability to stimulate antibody production in New Zealand White rabbits (Oryctolagus cuniculus). In addition, each animal was evaluated for adverse reactions. The antigen, rat liver microsomal epoxide hydrolase, was administered sc emulsified with FCA, TDM, or TDM + MPL. Serum antibody titers were stimulated with all 3 adjuvant-antigen combinations. The highest titer was produced by use of FCA; TDM + MPL produced an intermediate response, and TDM produced the lowest titer. All of the rabbits immunized with FCA developed sterile subcutaneous abscesses. Rabbits immunized with TDM or TDM + MPL developed no abscesses, and only slight reactions at the injection sites. The synthetic adjuvant system TDM + MPL is recommended for use in rabbits, considering its adequate stimulation of antibody production with minimal adverse reactions.

Ten colostrum-deprived calves were assigned to 1 of 2 treatment groups (5 calves/group), and fed colostrum that had either low (naturally infected cows) or high (immunized cows) antibody titers to bovine coronavirus (BCV). All calves were inoculated orally and intranasally with virulent BCV when they were 24 to 48 hours old and challenge exposed 21 days later. Blood, feces, nasal secretions, tears, saliva, and bronchoalveolar lavage (BAL) fluids were collected weekly from each calf for 5 weeks after inoculation. The titers to whole BCV or the relative amounts of isotype-specific antibodies to BCV structural proteins were evaluated in these samples by ELISA or immunoblotting, respectively. Both pools of colostrum contained primarily IgG1, IgG2, and IgA antibodies to the E2 and E3 BCV proteins. Calves fed the high-titer colostrum had correspondingly higher amounts of passive IgG1 and IgA antibodies to whole BCV and to the E2 and E3 BCV proteins in serum, feces, and BAL fluid at postinoculation week 1 than those calves fed low-titer colostrum. Active IgG1, IgA and IgM antibody responses in serum and active IgA and IgM antibody responses in most mucosal secretions to whole BCV and to the E2 and E3 proteins were lower or delayed in calves fed high-titer colostrum, compared with responses in calves fed low-titer colostrum. In contrast, increased responses to the BCV N protein were observed in all samples (except in serum and BAL fluid) in the calves fed high-titer colostrum, compared with calves fed low-titer colostrum. Upon challenge exposure, responses to E2 and E3 BCV proteins in serum and BAL fluid were lower in the group fed high-titer colostrum, compared with those in the group fed low-titer colostrum. Our findings indicate that the level of passive immunity in calves at the time of BCV inoculation can influence the development of active antibody responses in serum, feces, and mucosal secretions to whole BCV and to some BCV proteins individually.