A monoclonal antibody (MAB) against equine tumor necrosis factor-alpha (Eq TNF) was used to investigate the role of TNF in cytokine, eicosanoid, and metabolic responses of Miniature Horses given endotoxin. Plasma concentrations of interleukin 6 (IL-6), lactate, thromboxane A2 metabolite, and prostacyclin metabolite (6-keto-PGF(1 alpha)) were measured in 10 Miniature Horses given 0.25 micrograms of lipopolysaccharide (LPS; Escherichia coli O55:B5)/kg of body weight. Five horses were given Eq TNF MAB and 5 were given isotype-matched MAB as control. All horses were given 1.86 mg of antibody/kg by IV infusion, 5 minutes before LPS was given IV. Blood samples were taken 20 minutes before and at multiple intervals for 24 hours after LPS was given. Interleukin 6 bioactivity in plasma was measured, using IL-6-dependent cell line (B9). Eicosanoid activities were assessed by enzyme immunoassay, and plasma lactate concentration was determined enzymatically. Data were analyzed by ANOVA and Tukey's honest significant difference test for significant (P < 0.05) effect of treatment. Horses given Eq TNF MAB had significantly (P < 0.050) lower peak mean +/- SEM IL-6 (59 +/- 29 U/ml), lactate (16 +/- 2.00 mg/dl), and 6-keto-PGF(1 alpha) (254 +/- 79 pg/ml) values then did horses given control MAB (880 +/- 375 U/ml for IL-6; 26 +/- 0.04 mg/dl for lactate; and 985 +/- 290 pg/ml for 6-keto-PGF(1 alpha)). There was no effect of anti-TNF treatment on LPS-induced thromboxane A2 metabolite production. Tumor necrosis factor mediated IL-6, lactate, and prostacyclin responses, without affecting thromboxane production in horses given LPS.

Colostrum-deprived calves (n = 34) were fed various amounts of colostrum, colostrum substitute, or milk replacer to establish a range in titer of passively acquired viral neutralizing antibody in serum. The calves were then challenge exposed intranasally with a virulent, noncytopathic bovine viral diarrhea virus (BVDV-890). After viral challenge exposure, calves were monitored for fever, leukopenia, thrombocytopenia, and diarrhea. In addition, viral isolation and viral titration were performed on specimens of nasal secretions, buffy coat cells, and serum obtained from the calves. Fever and systemic spread of virus were detected in calves that had viral neutralizing titer of 256 or lower. Calves that had viral neutralizing titer lower than 16 developed severe clinical disease manifested by fever, leukopenia, thrombocytopenia, and diarrhea. Seventy and duration of signs of disease decreased as titers of passively acquired viral neutralizing antibody increased. These results indicate that low to intermediate titers of passively acquired viral neutralizing antibody were not sufficient to fully protect calves from virulent bovine viral diarrhea virus.

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.

Sequential histologic, immunologic, and virologic features of herpesvirus-induced keratitis were studied in 18 experimentally infected cats. Histologic changes were assessed by use of light microscopy, and the presence of viral antigen, B lymphocytes, and T lymphocytes was verified immunohistochemically. Flow cytometry was used to monitor changes in blood T lymphocytes (CD4 and CD8 homologues) and B lymphocytes. Cellular immunity was assessed by use of the lymphocyte proliferation assay. Development of stromal keratitis was preceded by prolonged absence of corneal epithelium, decreased numbers of circulating lymphocyte subsets, decreased mitogen responses, and acquisition of viral antigen by the corneal stroma. Return to normal of circulating lymphocyte numbers and function was temporally associated with the arrival of neutrophils and B and T lymphocytes in the corneal stroma. Sequelae to stromal inflammation were fibrosis and scarring. Findings suggest that suppression of local immune responses allows virus access to the corneal stroma, and that subsequent keratitis is mediated by an immune response to viral antigen.

Fifty serum samples from dogs with clinical signs of hypothyroidism and autoantibodies (AA) to thyroglobulin (Tg), thyroxine, or triiodothyronine were screened for AA to thyroid peroxidase (TPO). Thyroid peroxidase is the antigen against which microsomal AA are formed in human beings with lymphocytic thyroiditis. The TPO was isolated from canine thyroid tissue, using a modification of the procedure for purifying porcine TPO. The enzyme was solubilized from the membrane, using a deoxycholate-trypsin solution, followed by ammonium sulfate precipitation and diethylaminoethyl Sephadex chromatography. Activity of TPO was determined, using an iodide oxidation assay and a guaiacol assay. A monoclonal antibody to canine Tg, coupled to an immunoaffinity column, was used to eliminate the contaminating Tg from the TPO preparation. Using the TPO preparation as an antigen, an ELISA was performed on 10 serum samples and immunoblot assays were performed on 50 canine sera. Autoantibodies to TPO were not found in any of the sera. Assays also were performed, using purified porcine and human TPO and evidence of cross-reactivity with canine TPO was not identified. The absence of AA to TPO in dogs suggests a different pathogenesis for autoimmune thyroid disease in dogs than that hypothesized for lymphocytic thyroiditis in human beings.

We investigated the ability of an antibody-specific, O antigen-based ELISA to document Salmonella typhimurium herd infections by screening of milk samples. Three cattle populations, 20 herds with no history of salmonellosis, 8 herds with history of S. typhimurium episodes within the previous 7 months, and 220 herds of unknown disease status, were tested. A herd was considered ELISA positive if at least 5% of the cows had OD values > 0.3. Among the 20 herds without history of salmonellosis, only 2 herds were ELISA positive, whereas all 8 herds with a known history of salmonellosis were ELISA positive (herd specificity, 0.9 and herd sensitivity, 1.0). A significant correlation (P < 0.001) was found between the OD values of serum and milk samples from cows in the herds with a history of salmonellosis. It was concluded that ELISA testing of individual milk samples can be used for surveillance of herds for S. typhimurium infections, but further modifications are needed to test bulk tank milk samples.

Objective-To develop a flow cytometric assay for detection of platelet-bound IgG in dogs. Sample Population-Negative-control platelets were obtained from 5 clinically normal Greyhounds. Positive-control platelets were platelets from 1 clinically normal dog, sensitized with dog anti-canine platelet alloantibodies. Procedure-Washed platelets were incubated with mouse anti-canine IgG conjugated to fluorescein isothiocyanate and analyzed by flow cytometry. Optimal dilution of antibody reagent and dose-response were determined, as were effects on platelet-bound IgG detection of storage time and temperature of K3EDTA-anticoagulated blood samples, variable platelet numbers, and variable filling of K3EDTA evacuated tubes. Results-A 1:128 dilution of antibody reagent was optimal. There was a linear increase in platelet-bound IgG when normal canine platelets were incubated with increasing concentrations of positive-control serum. Variable numbers of positive-control platelets tested and variable filling of K3EDTA evacuated tubes had no significant effect on platelet-bound IgG concentration. Platelet-bound IgG concentration increased with storage time at room temperature (P = 0.0003), but not when blood was kept cool. Sufficient platelets for assay were able to be isolated from 3 ml of blood from 5 dogs with < 10,000 platelets/microliter. Conclusion-This assay for platelet-bound IgG in dogs is simple, repeatable, and practical. The assay is not affected by platelet count or variable filling of evacuated tubes, and requires only 3 ml of K3EDTA-anticoagulated blood. Blood samples for testing require packaging on ice and overnight delivery but, after arrival at the laboratory, can be refrigerated and analyzed within 72 hours of collection. Clinical Relevance-Assays for platelet-bound IgG may help in assessing causes and treatment of thrombocytopenia.

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.

Tumor necrosis factor-alpha (TNF) is an important mediator of endotoxin-induced pathologic changes. To help define the role of TNF in equids with endotoxemia, the effects of pretreatment with a murine monoclonal antibody (MAB) against equine TNF were evaluated in Miniature Horses given endotoxin. Five horses were given TNF MAB at a dosage of 1.86 mg/kg of body weight, IV, and 5 were given control MAB. Five minutes later, lipopolysaccharide (LPS; Escherichia coli O55:B5), 0.25 micrograms/kg, was given to all horses by bolus IV infusion. Clinical signs of disease were monitored at intervals up to 24 hours after LPS infusion, and blood was taken for determination of WBC count, PCV, plasma total protein concentration, plasma TNF activity, and serum MAB concentration. Reduction of plasma TNF activity in anti-TNF-treated horses was highly significant (P < 0.001), compared with that in control horses. Horses given TNF MAB had significantly improved clinical abnormality score (P < 0.010), lower heart rate (P < 0.001), and higher WBC count (P < 0.001), compared with horses given control MAB. Rectal temperature, respiratory rate, PCV, and plasma total protein concentration were not significantly different between groups. Serum MAB concentration peaked at 68 micrograms/ml 30 minutes after the end of antibody infusion in both groups. Neutralization of LPS-induced TNF activity reduced the hematologic and clinical responses of horses given LPS IV.

The core protein and the transmembrane protein, encoded for the structural genes gag and env, respectively, of caprine arthritis-encephalitis virus were amplified by use of polymerase chain reaction, cloned into a pGEX-2T vector, and expressed in Escherichia coli as fusion proteins with the glutathione S-transferase at their C-terminus. The recombinant proteins were purified and evaluated by use of an ELISA. Sera from 269 goats were tested, and the results were compared with those obtained by use of immunoblot analysis. When results from both recombinant ELISA (r-ELISA) were compared, it appeared that the transmembrane glycoprotein was more immunoreactive than the core protein, because it was recognized by a higher percentage of sera from infected goats. When results of the 2 ELISA (p28 r-ELISA and p40 r-ELISA) were combined in parallel, they were comparable to those of the immunoblot test, with sensitivity of 100% and specificity of 98.3%. It was also found that use of both r-ELISA makes it possible to compare the variable immunoreactivity against gag and env viral antigens, which may be correlated with the disease state. The r-ELISA, using core and transmembrane proteins, appears to be highly sensitive and specific for detection of antibodies against caprine arthritis-encephalitis virus.