Passive protection provided by sows inoculated with the virulent Miller strain of transmissible gastroenteritis virus (TGEV), or the ISU-1 strain of porcine respiratory coronavirus (PRCV), or both was evaluated in nursing pigs challenge exposed with virulent TGEV. Four sows (group B) were inoculated with PRCV oronasally twice at 4 and 2 weeks before parturition; 1 sow (group C) was inoculated similarly, but in 2 subsequent pregnancies; and 2 sows (group D) were oronasally primed with PRCV at 4 weeks before parturition, and 2 weeks later were administered a booster inoculation of virulent TGEV. Two additional sows (group E) remained uninoculated and served as seronegative controls, and 1 sow (group A) that had been naturally infected with TGEV served as a seropositive control. The degree of passive immunity transferred by these sows to their litters was assessed by challenge exposing the pigs of sows in groups BE (only the second litter of group C) with virulent TGEV at 3 to 5 days of age. After challenge exposure, clinical signs of infection and mortality were noted and fecal and nasal shedding of virus was assessed by ELISA. The IgA, IgG, and IgM antibody titers to TGEV were quantified in colostrum and milk of the sows by use of an isotype-specific monoclonal antibody-capture ELISA, using biotinylated monoclonal antibodies against each porcine isotype as detecting reagents. A plaque-reduction assay was used to quantify neutralizing antibody titers in serum, colostrum, milk, and fractionated whey (IgG and IgA/IgM). In the sow naturally infected with TGEV (group A), there was a pronounced decrease in IgG antibody titers to TGEV in the transition from colostrum to milk, and IgA TGEV antibodies became predominant, with high titers maintained throughout lactation. The 4 group-B sows partially protected their pigs after TGEV challenge exposure; mean mortality was 67%, compared with 100% in pigs suckling the 2 TGEV seronegative control sows (group-E litters). Although IgA TGEV antibodies were detected in colostrum and milk of group-B sows, IgG TGEV antibodies were the most abundant. The sow of group C had a marked increase in IgA TGEV antibody titers in colostrum and milk after reinoculation with PRCV during the second pregnancy, before TGEV challenge exposure of the litter. Its pigs were passively protected to a high degree after TGEV challenge exposure (27% litter mortality). The sows in group D, primed with PRCV and boosted with TGEV, provided the best passive protection after TGEV challenge exposure of their pigs. Not only litter mortality (27%) but also morbidity was reduced, compared with those factors for the other challenge exposed litters, and the sows did not become ill. In these swine, the high degree of passive protection observed could not be associated with the presence of only IgA TGEV antibodies in the milk, but high IgM TGEV antibody titers also were detected in colostrum and milk. Results of this study suggest that PRCV-inoculated sows are able to partially protect their pigs from TGEV challenge exposure and, on the basis of preliminary data, the degree of protection may increase after multiple PRCV exposures or after secondary exposure to TGEV during pregnancy. Also, an IgA respiratory tract-mammary gland link may exist as evident by the low titer of IgA TGEV antibodies in the milk of PRCV-inoculated sows, but may not be as efficient in inducing lactogenic IgA immunity as is the gastrointestinal tract-mammary gland link.

Receptors for opsonins, such as complement component C3b (CR1) and immunoglobulins, Fc receptors, interact with adhesion glycoproteins in mediating immune functions. Defects in expression of the adhesion glycoproteins CD11/CD18 results in severely hampered in vitro and in vivo adherence-related functions of leukocytes. Little is known regarding the effect of leukocyte adhesion deficiency (LAD) on ligand binding and receptor expression. We investigated the binding and expression of CR1 and Fc receptors by bovine neutrophils isolated from dairy calves suffering from LAD, compared with clinically normal (hereafter referred to as normal) age-matched calves. Neutrophils were also assayed for endogenously bound IgG and IgM and for exogenous binding of C3b, IgG1, IgG2, IgM, and aggregated IgG (aIgG), using flow cytometry. Luminol-enhanced chemiluminescence (CL) production in response to IgG2 opsonized zymosan was studied, and specific inhibition of CL was used to determine the specificity of IgG2 binding. Activation of protein kinase C with phorbol myristate acetate was used to determine the effect of cellular activation on expression of CR1. A greater percentage of neutrophils from normal calves bound C3b than did neutrophils from LAD-affected calves. Receptor expression was similar. Activation with phorbol myristate acetate resulted in increased expression of CR1 on neutrophils from normal and LAD-affected calves, but expression was almost twofold greater on neutrophils from normal calves. There was no difference between LAD-affected and normal calves in percentage of neutrophils that bound endogenous IgG and IgM. A greater percentage of neutrophils from normal calves bound exogenous IgM than did neutrophils from LAD-affected calves. Receptor expression for aIgG was greater on neutrophils from LAD-affected calves than on those from normal calves. Luminol-enhanced CL of neutrophils in response to IgG2 opsonized zymosan was not different between LAD-affected and normal calves. Results indicate increased binding and expression of Fc receptors for aIgG and decreased binding and expression for C3b and IgM on neutrophils from calves with LAD. Leukocyte adhesion deficiency may be compounded by added defects in the expression and binding of receptors for opsonins, such as C3b and IgM.

A series of experiments was conducted to document tumor necrosis factor-alpha (TNF) activity in serum of swine after inoculation with Salmonella spp endotoxin and after oral or respiratory tract challenge exposure with live Salmonella spp. For experiment 1, a potentially lethal dose of S typhimurium endotoxin (25 microgram/kg of body weight) was administered IV, and serum TNF activity was measured. High TNF (approx 700 IU/ml) activity at 1 to 2 hours after administration of the inoculum was associated with death, whereas lower TNF (approx 30 IU/ml) activity was associated with a general prolonged state of shock. For experiment 2, pigs were administered a nonlethal dose (5 microgram/kg, IV) of either S typhimurium or S choleraesuis endotoxin. Difference in the ability to induce porcine serum TNF activity was not observed between strains. During experiment 3, pigs were inoculated with 104 colony-forming units of S typhimurium chi4232 either orally by gelatin capsule (GC) or by intranasal (IN) instillation. A late serum TNF response (17 IU/ml) was measured at 6 weeks after IN inoculation. A serum TNF response was not detected in GC-inoculated pigs. All tissues and feces were test-negative for S typhimurium prior to the 6-week TNF response. Serum TNF activity may be related to clearance of S typhimurium after respiratory tract exposure, but it is not important to or indicative of clearance of orally presented S typhimurium in swine. During experiment 4, pigs were inoculated with 106 colony-forming units of S typhimurium chi4232 similarly as for experiment 3. Challenge exposure with this medium-size dose of inoculum induced a prolonged peak serum TNF response (37 IU/ml) between 2 and 4 weeks after IN inoculation. Again, serum TNF activity was not detected in GC-inoculated pigs. Data suggest that clearance of a medium-size dose (106) of inoculum may be influenced by the prolonged higher serum TNF activity. For experiments 5 and 6, pigs were inoculated IN with 103, 106, 108, or 109 S choleraesuis chi3246. A measurable, yet statistically nonsignificant, serum TNF response was observed for all doses. Pigs inoculated by GC with 108 S choleraesuis chi3246 had similar results. High does (> 106) of live S choleraesuis were associated with clinical signs of endotoxic shock. Clearance of S choleraesuis, or lack thereof, did not correlate with serum TNF activity.

Investigation of the structure of equine articular cartilage link protein (LP) from individuals ranging in age from 1 to 15 years identified 3 distinct isoforms having molecular weights of 46,000, 43,000, and 41,000. The relative amounts of each of the 3 isoforms altered with age. The largest form did not change with age; however, amounts of the Mr 43,000 and 41,000 forms increased with increasing age. The results suggested that an accumulation, in the extracellular matrix of cartilage, of these 2 smaller products may have arisen from proteolytic cleavage. The complete amino acid sequence of the protein core was determined from complementary DNA products prepared by polymerase chain reaction amplification of cartilage LP mRNA. The sequence had 96% similarity with human LP and with that of other species for which the primary structure has been determined. This high degree of sequence conservation and the isoform data indicate that extracellular processing of LP occurs by similar mechanisms in various species. At the transcription level, equine chondrocytes were found to express LP as 2 abundant mRNA of 5.0 and 3.0 kb, and a smaller mRNA of 1.5 kb. Processing of the LP mRNA in horses, thus, appears to be similar to that found in other species investigated, and although multiple transcripts are present, the coding region remains unaltered and only 1 protein product is made.

Intraocular production of Toxoplasma gondii-specific antibody in cats has been estimated by comparing the ratio of T gondii-specific antibody in aqueous humor and serum with the ratio of total immunoglobulins in serum and aqueous humor (Goldmann-Witmer coefficient; aqueous antibody coefficient; C value). It has been proposed that in human beings, comparison of the ratio of T gondii-specific antibody in aqueous humor and serum with the ratio of antibodies against a nonocular pathogen in serum and aqueous humor is more accurate than methods using total immunoglobulin quantification. We developed an ELISA for detection of calicivirus-specific antibodies in the serum and aqueous humor of cats. By evaluating calicivirus-specific antibody concentrations in the aqueous humor of healthy and diseased cats, calicivirus was assessed as a nonintraocular pathogen. The ratio of T gondii-specific antibodies in the aqueous humor and serum and the ratio of calicivirus-specific antibodies in serum and aqueous humor were evaluated as a means of estimating intraocular T gondii-specific antibody production. A field strain of feline calicivirus was isolated, cultured, and purified. A calicivirus-specific IgG ELISA was developed for detection of feline calicivirus-specific IgG in serum and aqueous humor. Calicivirus-specific IgG was measured in the serum and aqueous humor from 3 groups of control cats. Results suggested that calicivirus is a nonintraocular pathogen in cats and that calicivirus IgG detected in aqueous humor is attributable to leakage across a damaged blood-ocular barrier. Intraocular production of T gondii-specific antibodies was estimated, using 2 formulas. The C value was calculated by multiplying the ratio of T gondii-specific IgM or IgG in aqueous humor and serum by the ratio of total immunoglobulins (using the corresponding IgM or IgG class) in serum and aqueous humor. The Ctc value (Toxoplasma-calicivirus Goldmann-Witmer coefficient) was calculated by multiplying the ratio of T gondii-specific IgM or IgG in aqueous humor and serum by the ratio of calicivirus-specific IgG in serum and aqueous humor. Serum and aqueous humor samples were obtained from 41 client-owned cats with uveitis, and T gondii-specific C values and Ctc values were calculated. Toxoplasma gondii-specific IgM or IgG C values of 10 or greater or T gondii-specific IgM or IgG Ctc values of 1 or greater were considered to be suggestive of intraocular T gondii-specific antibody production. Of the 41 cats, 20 (48.7%) had evidence of intraocular production of T gondii-specific antibody on the basis of either an IgM or IgG C value of 10 or greater. A Ctc value could not be calculated in 3 cats because calicivirus-specific IgG was not present in aqueous humor. Of the 38 cats for which Ctc values could be calculated, 25 (65.8%) had evidence of intraocular production of T gondii-specific antibody on the basis of either an IgM or IgG Ctc value of 1 or greater. The C values and Ctc values were in agreement for 75.9% of IgM containing samples and 75% of IgG containing samples. Sensitivity, specificity, predictive value of a positive test result, and predictive value of a negative test result for an IgM or IgG C value, when compared with the corresponding IgM or IgG Ctc value were determined. The results indicate that use of the C value for estimation of intraocular T gondii-specific antibody production will result in 28.6 (IgM) to 50% IgG) false-negative results and 12.5% (IgM and IgG) false-positive results, when compared with the Ctc value.

Administration of an N-lauroylsarcosine-derived outer membrane protein fraction of Pasteurella haemolytica A1 (SCI-1) induced a protective response in calves against intrathoracic challenge exposure with the homologous serovar. Outer membrane proteins from heterologous serovars, A6 and A9, induced partial protection that was associated with their respective similarities to serovar A1 in outer membrane protein profiles derived by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Calves vaccinated with SCI preparations did not have detectable neutralizing antibody to P haemolytica A1 leukotoxin. Antibodies to whole-cell antigens, carbohydrate-protein subunit antigen, and SCI-1 were associated with resistance, which indicates that protein antigens shared among cell surface, carbohydrate-protein subunit, and SCI preparations are immunogenic and enhance resistance to experimental challenge exposure.

Thirty-five heifers were allotted to 3 groups. Group 1 (contro]) consisted of 10 heifers that were not vaccinated and were challenge exposed by breeding to infected bulls. Group 7 (natural challenge exposure) consisted of 10 heifers that were vaccinated and challenge exposed by breeding to infected bulls. Group 3 (experimental challenge exposure) consisted of 15 heifers that were vaccinated and challenge exposed by breeding to infected bulls and by intravaginal inoculation with 10(7) Tritrichomonas foetus. Total immunoglobulin concentrations and specific trichomonal antibodies were determined in serum and vaginal secretions of heifers, using radial immunodiffusion and ELISA procedures. Control heifers remained infected for a mean of 10.6 weeks (range, 0 to 18 weeks), and heifers of the natural and experimental challenge-exposure groups remained infected for 3.2 and 5.0 weeks, respectively (range, 0 to 12 weeks). Total serum and cervicovaginal mucus concentrations of IgM, IgA, IgG1, and IgG2 did not change significantly after vaccination or challenge exposure. However, ELISA titers of total trichomonal antibodies increased up to 1:10,000 (range, 1:400 to 1:10,000) in serum after vaccination, and increased approximately tenfold above background in cervicovaginal mucus. In serum, the predominant trichomonal antibody isotype was IgG1, although trichomonal IgA and IgM antibodies also increased. The predominant trichomonal antibody detected in cervicovaginal mucus was IgA. Antibody titers in serum and cervicovaginal mucus of vaccinated heifers were not increased by infection. However, in control heifers, the total local trichomonal antibody response increased three- to fivefold after infection. In these heifers, specific antibodies in serum were predominantly IgG1 and local (cervicovaginal) antibodies were predominantly IgA.

The effects of 3 days of glucocorticoid administration on bovine blood neutrophil expression of L-selectin and CD18, and on the health status of mammary glands subclinically infected with Staphylococcus aureus were measured in 9 lactating Holsteins. The experiment was a 3 x 3 Latin square cross-over design, with 3 glucocorticoid treatments switched among groups of 3 cows/treatment during 3 periods. Treatments consisted of a vehicle (control, 10 ml of excipient/cow/d), cortisol (7.5, 15, and 7.5 mg/cow on days 1, 2, and 3, respectively), and dexamethasone (0.04 mg/kg of body weight/cow/d for total daily dosages that ranged from 21.6 to 33.2 mg). Blood samples for immunostaining and flow cytometric analysis of L-selectin and CD18 and leukograms, as well as foremilk samples for determination of S aureus shedding somatic cell counts, protein and fat percentages, and daily milk yields were collected repeatedly before, during and after treatment days. Dexamethasone caused a profound, acute, short-lived down-regulation of L-selectin on neutrophils, which correlated in time to leukocytosis, mature and immature neutrophilias, increased shedding of S aureus in infected glands, and onset of high percentages of fat and protein and decreased milk yields. Dexamethasone also caused profound but delayed down-regulation of neutrophil CD18, which reached nadir simultaneously with reappearance of L-selectin-bearing neutrophils, normalized blood neutrophil counts, markedly high foremilk somatic cell counts and protein percentage, decreased S aureus shedding in milk, and finally, expression of clinical mastitis in some infected quarters. Each of these variables had returned to control (vehicle) values by the ninth (and last) sample collection day. Although cortisol treatment also decreased expression of L-selectin and CD18 on neutrophils, dosages used in this study were not sufficient to alter the number of circulating cells or to convert subclinical mammary gland infections to clinical mastitis. These results suggest that mammary gland health status can be altered by sudden exposure of blood neutrophils to glucocorticoids, because these steroid hormones caused profound down-regulation of the adhesion molecules that direct neutrophil margination and migration through the vascular endothelium. The results also reinforce the potential disease risk of treating infected animals with potent synthetic glucocorticoids, such as dexamethasone.

Systemic administration of dexamethasone led to a significant reduction in the size of the tuberculin reaction in response to intradermal injection of bovine purified protein derivative in 18 cattle experimentally sensitized to Mycobacterium bovis (P < 0.01) and 8 cattle naturally infected with M bovis (P < 0.001). The reaction in 6 of the 7 M bovis-infected cattle that received dexamethasone was classified as negative for the standard interpretation of the single intradermal comparative tuberculin test. Significantly fewer BoCD2+ (P < 0.05) and BoCD4+ T cells (P < 0.001) were present at the reaction site and in blood of dexamethasone-treated cattle, compared with untreated control cattle. Significantly fewer cells expressing the interleukin-2 receptor and WC1+ gamma delta T cells (P < 0.001), and a significantly greater number of cells expressing the ACT2 antigen (P < 0.05) were found at the reaction site in dexamethasone-treated cattle than in controls. The number of BoCD8+ T cells at the reaction site and in blood was not significantly affected by administration of dexamethasone. In vitro production of interferon-gamma by lymphocytes incubated with bovine purified protein derivative also was significantly lower (P < 0.01) in the dexamethasone-treated cattle.

Marked differences in bone marrow cellularity were observed between cattle affected with leukocyte adhesion deficiency (LAD) and control cattle. The number of nucleated cells in bone marrow was 2.9 to 8.8 times higher in cattle affected with LAD, compared with controls. The myeloid-to-erythroid ratio of bone marrow from 3 cattle affected with LAD ranged from 2.4 to 12. Deficient CD18 expression on neutrophils isolated from bone marrow of cattle with LAD was clearly detected by flow cytometric analysis. Neutrophils from bone marrow of cattle affected with LAD appeared round and not flat, after adherence to plastic wells under agarose, whereas neutrophils from bone marrow of clinically normal cattle were firmly spread on the surface of plastic wells. In the chemotaxis under-agarose assay, many pseudopodia were detected on bone marrow neutrophils from clinically normal cattle, but were not detected on bone marrow neutrophils from cattle with LAD. Activities of chemotactic movements and phagocytosis of neutrophils isolated from bone marrow of cattle affected with LAD were documented to be severely impaired.