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.

We used monoclonal antibodies and immunohistologic examination of lymph nodes, to elucidate the pathogenesis of lymphosarcoma induced by, infection with bovine leukemia virus (BLV). The superficial cervical lymph nodes from 3 BLV-infected but apparently healthy sheep and 5 sheep with full-blown lymphosarcoma were examined. We also investigated the integration of bovine leukemia provirus by use of Southern blotting. In lymph nodes from sheep lacking clinical signs of infection, in which the provirus had been integrated at multiple sites in the genome, many large hypertrophic follicles were observed in the cortex. These follicles had germinal centers consisting of CD4+T cells and B cells that expressed surface IgM (sIgM) and major histocompatibility complex (MHC) class-II antigens, but not B cell-specific B2 molecule. The percentage of CD4+T cells in the cortex was significantly (P < 0.05) higher than that of the controls and sheep with lymphosarcoma. In all sheep with lymphosarcoma, the lymph nodes were completely destroyed by proliferating neoplastic cells, and in addition, small atrophic follicles, which consisted of normal B-cell marker-positive cells, were seen near the trabecula and the subcapsule. In these instances, neoplastic cells appeared to be a monoclonal population derived from a single CD5- B-cell lineage and to be classified as 2 types, CD5-CD4-CD8-B2+MHC class-II+sIgM+ and CD5-CD4-CD8-B2+MHC class-II+sIgM-. Moreover, CD8+T cells infiltrated diffusely throughout the tumorous lymph nodes apart from the atrophic follicles, and CD4+T cells were observed around atrophic follicles. Both types of T cells were small-size, normal lymphocytes with round and noncleaved nuclei, and were apparently non-neoplastic cells. In fact, after separation by use of a panning method, it seems that, in blood mononuclear cells from BLV-infected sheep without clinical signs of infection, but in lymphosarcomatous stages, the proviral genome was integrated only in B cells and not in T cells. Thus, we conclude that the host's immune response may be still maintained at a lymphosarcomatous stage.

A method for quantification of serum total IgE concentration in dogs by use of an ELISA containing monoclonal mouse anti-canine IgE was developed. Microtitration plates were coated with monoclonal mouse anti-canine IgE. Test sera and reference serum dilutions were added, followed by biotinylated monoclonal mouse anti-canine IgE. Avidin-alkaline phosphatase conjugate was added, and color development was measured spectrophotometrically, using a microtitration plate reader. Quantitative results were obtained by assigning to a reference serum a value of 100 IgE units/ml. Absorbance values of unknown samples were converted into IgE units by comparison with a standard curve generated by measurement of reference serum dilutions. Intra- and interassay coefficients of variation were 5 and 7%, respectively, and assay sensitivity was 1 U/ml. The assay was used to establish a normal range for total IgE concentrations in 30 healthy dogs. Total IgE concentration in healthy dogs followed a skewed distribution and ranged from < 1 to 91.2 U/ml, with a geometric mean value of 7.1 U/ml. The IgE concentration was remarkably stable in serum samples subjected to 25 freeze/ thaw cycles or incubation at approximately 25 C (room temperature) for up to 10 days. Comparison of total IgE concentrations in 23 serum samples assayed by use of double-overlay radial immunodiffusion and ELISA yielded correlation coefficient of 0.94. Comparison of the reference serum standard curve with serial dilutions of a purified IgE solution of known concentration yielded a range of values for the IgE unit of 0.7 to 2.0 micrograms.

Effect of estrogen (E2) and progesterone (P4) on uterine antibacterial activity and immunoglobulin concentrations in mares was studied. In 2 in vitro experiments, 6 mixed-breed mares were ovariectomized, and uterine fluid and blood serum were analyzed. Antibacterial assay methods were used to determine inhibitory effects on Streptococcus zooepidemicus of uterine fluid samples collected on days 3, 5, and 8, and serum obtained on day 8 of treatment. Single radial immunodiffusion methods were used to quantify amounts of IgA and IgG in uterine fluid and serum on days 3, 5, 8, and 14 of treatment. Neither E2 nor P4 increased activity of serum and uterine fluid against S zooepidemicus. Numbers of colony-forming units per milliliter of bacteria were significantly (P < 0.01) lower in control Hanks' balanced salt solution with 1.0% gelatin (HBSSG) than in uterine fluids. Bacterial numbers were significantly (50%) greater in uterine fluids and serum than in HBSSG controls for both treatments. Both fluids, especially serum, supported significantly (P < 0.01) more growth of S zooepidemicus than did HBSSG when incubated for 0, 2, and 4 hours. These findings are in contrast to previous reports of antibacterial activity in the uterus of sexually intact mares undergoing an estrous cycle: great reduction of bacterial count in uterine fluid from mares in diestrus, and significant increases in bacterial numbers in uterine fluid or serum from mares in estrus. Treatment comparisons between serum and uterine fluid IgA and IgG concentrations were not significantly different, although overall IgA concentration in the uterus was higher than concentration in serum. The IgG concentration in uterine fluid was higher in P4- than E2-treated mares. However, IgG concentration was significantly (P < 0.01) higher in uterine fluid on day 8 in P4-treated mares than on day 3 or 5. Results of this study indicate that neither immunoglobulin concentration nor hormone treatment has a direct effect on streptocidal activity.

Cell-mediated immunity was evaluated in intestinal, respiratory, and systemic lymphoid tissues of pigs exposed when 11 days old to virulent transmissible gastroenteritis virus (TGEV), attenuated TGEV, or porcine respiratory coronavirus (PRCV), 3 antigenically related porcine coronaviruses with distinct enteric and respiratory tissue tropisms. Mononuclear cells were prepared from mesenteric lymph nodes (MLN), bronchial lymph nodes (BLN), and spleens of pigs and tested for virus-specific responses by use of lymphocyte proliferation assays. Vigorous MLN and BLN proliferation responses to virulent TGEV and PRCV, respectively, at postinoculation days 8 to 24 were strongly associated with prior detection of TGEV in rectal swab samples and PRCV in nasal swab samples. Gastrointestinal disease and intestinal virus replication, assessed on the basis of rectal virus shedding, were almost exclusively found in the virulent TGEV-inoculated pigs, even though virulent TGEV and a high dose of attenuated TGEV elicited the highest proliferation responses in MLN. Pigs exposed to PRCV or attenuated TGEV did not have clinical signs of disease, and only 1 pig given a high dose of attenuated TGEV shed virus in feces. Porcine respiratory coronavirus replicated in the respiratory tract after either oronasal or aerosol inoculation of virus and induced strong BLN, but not MLN, proliferation responses. A high dose of attenuated TGEV (4 X 10(8) plaque-forming units) was more effective than a lower dose of attenuated TGEV (7 X 10(6) plaque-forming units) in eliciting significant lymphocyte proliferation in MLN and BLN. Cellular immune function, assessed on the basis of mitogen-induced proliferation of lymphocytes, was comparable for all 3 sources of lymphocytes and was not adversely affected by exposure to any of the pigs. The tissue tropism of TGEV and PRCV was associated with induction of virus-specific cell-mediated immune responses, as evidenced by substantial lymphocyte proliferation responses in MLN and BLN, mucosa-associated lymph nodes adjacent to the primary sites of virus replication. The failure of PRCV strain ISU-1 to replicate in the intestinal tract correlated with poor virus-specific cellular immune responses in MLN.

Effects of dietary ochratoxin A (OA) and T-2 toxin, fed singly and in combination, were evaluated in growing crossbred pigs. Thirty-six barrows (3 replicates of 3 for each of 4 treatment groups, mean body weight, 18.0 kg) were fed: 0 mg of OA and 0 mg of T-2/kg of feed (control); 2.5 mg of OA/kg of feed; 8.0 mg of T-2/kg of feed; or 2.5 mg of OA plus 8.0 mg of T-2/kg of feed for 30 days. Production performance, serum biochemical, hematologic, immunologic, and pathologic evaluations were made. Body weight and body weight gain were decreased by all toxin treatments, but the combination toxin treatment reduced weight gain more than did either of the toxins administered singly and could be considered additive. Liver weight was decreased by combination treatment, whereas kidney weight was increased by OA treatment. Ochratoxin decreased serum cholesterol, inorganic phosphorus, and alkaline phosphatase values; reduced mean cell volume, hemoglobin concentration, and macrophage phagocytosis; and increased creatinine and total protein values. Consumption of T-2 toxin reduced hemoglobin and serum alkaline phosphatase values. The combination treatment decreased serum cholesterol, gamma-glutamyltransferase, alkaline phosphatase, mean cell volume, hematocrit, and hemoglobin values, as well as lymphoblastogenesis and phagocytosis, and increased serum nine concentration. We concluded that OA and T-2, singly or in combination, can affect clinical performance, serum biochemical, hematologic, and immunologic values, and organ weights of growing barrows. Although some analytes were affected more by the combination than by either toxin alone, the interactions could best be described as additive, not synergistic.

The main objective of the study reported here was to generate a panel of monoclonal antibodies (MAB) to bovine neutrophil surface antigens, and to identify MAB that modulate neutrophil chemotaxis, respiratory burst, and phagocytosis. A further objective was to study MAB reactivity with resting and activated neutrophils, to identify activation antigens and adhesion molecules. A panel of 14 MAB was generated by producing murine hybridomas. Neutrophils incubated with MAB at 4 C for 2 hours were used in chemotaxis, respiratory burst, and phagocytosis assays. Chemotaxis was evaluated in Boyden chambers, using Escherichia coli endotoxin-activated fetal bovine serum as the chemoattractant. Respiratory burst was determined by measuring chemoluminescence of neutrophils incubated with 5-amino-2,3-dihydro-1,4-phthalazinedione, and serum opsonized zymosan. Phagocytosis was determined by flow cytometry, using fluorescein-labeled Staphylococcus aureus. The MAB S7G8, S5F8G10, S7E10, and S5F8B8 enhanced chemotaxis (to > 125% of control). The MAB S7E10 and S8D9 enhanced respiratory burst activity (to > 125% of control), whereas MAB S2G8, S4G10, S8G10, and S5F8B8 caused inhibition (to < 75% of control). The MAB S2G8, S4G10, S8G10, and S5F8G10 enhanced phagocytosis (to > 125% of control). Chemotaxis, respiratory burst, and phagocytosis values of neutrophils not bound with MAB served as controls for comparison. The MAB binding for nonactivated neutrophils (at 4 C) ranged from 9 to 100%, and for activated neutrophils (at 37 C; at 37 C with phorbol myristate acetate) from 90 to 100%. Binding of MAB S4F5, S5F8B8, S6C6, S7E10, S8D9, and S5F8G10 increased when neutrophils had been incubated at 37 C. Binding of these MAB was further increased after incubation with phorbol myristate acetate (100 ng/ml) at 37 C, indicating recognition of activation antigens by MAB. The MAB generated in this study appeared to be potential candidates for studying mechanisms of neutrophil function and for enhancing neutrophil function in vitro and in vivo.

Hemagglutinins HA) of H1N1 swine influenza viruses isolated in the United States have remained antigenically and genetically conserved for many years. In contrast to such conservation, the RA of A/Swine/Nebraska/1/92 (Sw/Neb) could readily be distinguished from those of contemporary porcine viruses. Twenty-eight amino acid mutations differentiated the HA of Sw/Neb and A/Swine/Indiana/1726/88, the most recent H1N1 swine influenza virus for which HA sequence data were available. Among these differences were mutations at potential asparagine-linked glycosylation sites and charge changes at many residues. The Sw/Neb virus also could be differentiated from other swine influenza viruses in hemagglutination-inhibition assays with monoclonal antibodies to recent H1 swine HA. Nonetheless, overall sequence analysis of the HA and the nucleoprotein genes of Sw/Neb indicated that this virus was more closely related genetically to classic H1N1 swine influenza viruses than to H1N1 avian or human viruses. Infection of swine with Sw/Neb under experimental conditions induced clinical signs and lesions typical of swine influenza. However, affected swine in the field had high, persistent fevers, but relatively mild signs of respiratory tract disease. This study indicated that an antigenically and genetically novel variant of swine influenza virus was detected in the United States.

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.

Cows and calves from a 1,600-cow drylot dairy were screened for IgG antibodies to Salmonella dublin lipopolysaccharide (LPS), using an indirect ELISA. The ELISA was performed on milk samples from lactating cows and on sera from nonlactating cows and calves. Fecal samples were collected from calves and nonlactating cows for culture of Salmonella spp. All seropositive cattle were retested by culture and ELISA 5 times at monthly intervals or until antibody concentration decreased. None of the cattle remained culture-positive and seronegative. Prior to and during the sample collection period, approximately 30% of calves < 8 weeks old died of S dublin infection. Vaccination of cows with a killed S dublin/S typhimurium vaccine at cessation of lactation was a routine management practice. The ELISA-determined Igg response to vaccination had decreased by 50 days after vaccination. Eight cows and 5 calves that maintained a high serologic response to S dublin were purchased and moved to a research facility for 6 months of intensive monitoring. Lactating cows were milked twice daily, and culture of milk and feces for Salmonella spp was performed 5 times/wk. Serum IgG antibodies to S dublin LPS were measured weekly, using ELISA. At the end of 6 months, all 13 cattle were necropsied and tissues were obtained for culture of Salmonella spp. All 8 cows and 5 calves maintained persistently high ELISA titer for the 6 months of testing, and shed S dublin in the milk and/or feces during the same period. On this basis, they were termed S dublin carriers. Salmonella dublin was isolated from mammary tissue of 2 calves at necropsy, indicating that bacteremia may be a mode of mammary infection by S dublin. Results of the study indicated serologic testing can be used successfully on a large dairy to identify S dublin carrier cattle. Using initial milk screening, 42 of 1,268 lactating cows were identified as suspect, requiring repeated serologic testing. One nonlactating cow, 7 of the 42 suspect lactating cows, and 5 of the 222 calves maintained an Igg response, and were found to be S dublin carriers. Carrier cows shed S dublin in 3.35% of fecal samples and 2.51% of milk samples, and carrier calves shed S dublin in 17.26% of fecal samples.