Objectives of this investigation were to extract and isolate protein fractions inhibitory to the cytotoxic properties of tumor necrosis factor-alpha (TNF-alpha). In this context, mixed populations of WBC were harvested from equine blood and were stimulated with a combination of a synthetic chemotactic peptide and a calcium ionophore. Several methods were subsequently applied for the initial preparation of cell-free crude protein extracts, including fractional precipitation with gradient concentrations of ammonium sulfate and preparative-scale isoelectric focusing. In addition, protein fractions were harvested from extracts of concentrated equine urine. Protein extracts of urinary origin were further separated by gel-filtration column chromatography. Identification of protein fractions possessing properties inhibitory to the cytotoxic characteristics of TNF-alpha was facilitated by a tissue culture-based technique for the biological assay of TNF-alpha-mediated cytotoxicity. Purified protein extracts possessed a marked ability to inhibit or neutralize the cytotoxic properties of TNF-alpha on the basis of survival of murine fibrosarcoma cell populations, compared with appropriate negative and positive reference controls. Relative purity of inhibitors and estimation of approximate molecular weight were established by conventional reducing and nonreducing sodium dodecyl sulfate. polyacrylamide gel electrophoresis analysis. Equine inhibitory protein fractions from mixed WBC populations, purified in the manner described, had molecular weights of 70,000 to 80,000 and 28,000. An analogous protein fraction of 28 kDa also was isolated from equine concentrated urine. Estimated isoelectric point of TNF-alpha inhibitor protein fractions was between pH of 5.5 and 6.1. These physical characteristics of equine TNF-alpha inhibitor protein fractions were similar to those described for a membrane-associated TNF-alpha receptor protein shed from chemotaxin- and calcium-ionophor-stimulated human WBC populations.

A highly sensitive enzyme-linked immunoelectrotransfer blot (EITB) assay, capable of detecting aphthovirus-specific antibodies to replicating virus in sera from cattle with persistent infection, was developed. The assay uses a set of purified recombinant DNA-derived nonstructural viral antigens as serologic probes in lieu of the traditionally used virus infection-associated antigen(s) partially purified from baby hamster kidney-infected cells. Sera from cattle with experimentally induced aphthovirus infection were analyzed sequentially by EITB at various postinoculation days, and the results were compared with those obtained by currently used techniques. It was established that, in aU cases, EITB results remained positive at late stages of infection. At these times, results of virus infection-associated antigen-antibody determinations were negative by use of the conventional immunodiffusion in agarose gel test, and virus was recovered only occasionally from esophageal-pharyngeal fluid. Specificity of the EITB test was indicated by negative results for sera from cattle in aphthovirus-free areas, including samples from cattle infected with a variety of bovine viruses. Moreover, the test eliminated a substantial number of false-positive results (on the basis of the immunodiffusion in agarose gel assay) caused by reactivity of sera from vaccinated cattle. Use of additional nonstructural viral antigens, other than RNA polymerase, is proposed to differentiate between seropositivity resulting from vaccination or infection. This procedure may be considered to have potential applications as a sensitive, safe, rapid, and economic field test for specific diagnosis of persistent aphthovirus infection in affected animals.

We used size-exclusion high-performance liquid chromatography (HPLC) to investigate the properties of the 2 isoforms of vitamin A-containing (holo) retinol-binding protein (RBP) in animals: the form that is bound to transthyretin (holo-TTR-RBP), and the form that does not bind to TTR (holo-free RBP). We also used radial immunodiffusion to measure immunologically active RBP (apo+ holo RBP). We compared the isoforms of RBP in animals with those of human beings to determine which animal is the best model of human RBP. Size-exclusion HPLC detected holo-free and holo-TTR-RBP in every animal species studied. Apparent concentration of holo-TTR-RBP varied among species: that of rabbits and dogs much greater than that of apes, sheep, goats, monkeys, rhinoceroses, felids, rats, human beings, and deer greater than that of pigs, zebra, and bison greater than that of penguins. Dogs have unusual RBP chromatograms; they have high concentration of RBP, but also appear to transport much of their vitamin A on proteins other than RBP, Human RBP antibody preparations could detect apo + holo RBP immunologic activity only in apes, monkeys, and felids. Apes and monkeys appeared to have complete cross-reactivity to human RBP antibodies. Felids may have substantial, but partial, cross-reactivity. Apes and monkeys appear to be the most relevant animal models for study of human RBP transport. However, there is a need for less-expensive models. Further research is needed, but in the interim, rats or sheep may be satisfactory for some purposes.

An ELISA, using hot saline solution extracts (HSS) of a less-mucoid variant (M -) strain of Brucella canis as antigen, was developed for detection of antibodies against B canis in dogs. The test was applied to 177 field serum samples previously tested by use of the 2-mercaptoethanol rapid slide agglutination test, 2- mercaptoethanol-tube agglutination test, and agar gel -immunodiffusion containing HSS and cytoplasmic antigens of B canis. Results indicated that this ELISA seems to be highly specific (95.6%) and slightly less sensitive (93.8%). The HSS obtained from B canis wild-type RM 6/66 also have been used, but in our study, it seemed to be unsuitable for use in ELISA because of the high background values observed for sera with negative test results.

Saline (0.9% NaCl) solution, flunixin meglumine (1.1 mg/kg), prednisolone sodium succinate (1.1 mg/kg), U74389F (1.5 mg/kg), and dimethyl sulfoxide (0.5 g/kg) were each administered IV to 5 neonatal calves 15 minutes after the start of a 3-hour infusion of Escherichia coli lipopolysaccharide (LPS; 2 micrograms/kg/ hr). Four additional calves were given a 3-hour IV infusion of saline solution alone. Only flunixin significantly suppressed eicosanoid production and mitigated clinical signs associated with endotoxemia. Prednisolone provided partial protection against LPS-induced hypotension and lacticemia. Pronounced hypoglycemia and lacticemia were observed in U74389F-treated calves; LPS-induced hypotension and hypoglycemia were marked in dimethyl sulfoxide-treated calves.

Seven mature dairy cows from 6 herds were obtained with history, clinical signs of disease, and laboratory findings suggestive of advanced paratuberculosis. A surgically implanted collection chamber was used to obtain peripheral tissue fluid. Blood, mammary gland flush fluid, and collection chamber flush fluid (CCFF) samples were obtained 6 times over a 2-week period from each cow. Mononuclear cell-rich portions of these fluids obtained by gradient centrifugation were submitted for bacteriologic culture of Mycobacterium paratuberculosis and for total and differential cell counts. Bacteriologic culture of feces for M paratuberculosis and complete necropsy performed on each cow at the conclusion of the study confirmed the diagnosis of paratuberculosis. Numbers of tissue macrophages obtained from CCFF samples were lower than expected. Mean (+/- SD) differential count of tissue macrophages collected from CCFF was 65.57 (+/- 23.39). Mean calculated tissue macrophages (total cell count X differential count) collected from CCFF samples was 623.1 (+/- 784.55) cells/microliter. Mycobacterium paratuberculosis was isolated from 1 of 42 (2.4%) collections of mononuclear cell-rich portions of plasma and from 2 of 42 (4.8%) CCFF samples. Mycobacterium paratuberculosis was not isolated from any collections of mammary gland flush fluid. The collection and processing techniques used in this study did not enhance detection of M paratuberculosis infection in cows with advanced paratuberculosis, beyond that of ileocecal lymph node biopsy or fecal culture.

Replication of bluetongue virus (BTV) in leukocytes from the blood of sheep, cattle, elk, and mule deer inoculated with BTV serotype 10 or 17 was assessed by immunocytochemical staining and dot blot northern hybridization to determine if differences in the prevalence of infection in this blood fraction might account for the differences in clinical disease among these species. Viremia was confirmed by virus isolation in all inoculated animals. Analysis of leukocytes with monoclonal antibodies specific for BTV proteins revealed low numbers of infected leukocytes in only 2 sheep 8 days after inoculation with BTV serotype 10. Most of the cells expressing BTV were identified morphologically as monocytes; approximately 10% of infected cells were lymphocytes. Bluetongue virus was not detected by use of dot-blot hybridization on samples of blood. Our results suggest that differential infection of leukocytes does not account for the pronounced differences in clinical signs and pathologic changes among ruminants.

Tissue variation in microscope slides made for spermatozoon analysis and variation introduced by the subjective techniques used to analyze these slides reduce the statistical power of studies that seek to use spermatozoon morphology to predict fertility. A simple specimen preparation method was developed to standardize stallion spermatozoon morphologic smears, and a new, automated spermatozoa morphometry instrument was used to objectively analyze the efficacy of the specimen preparation technique. The method achieved a standard spermatozoon concentration and reduced field-to-field variation in the number of spermatozoa analyzed. Metric measurements of spermatozoon head dimensions from clinically normal, fertile stallions revealed small, but highly significant, differences between stallions. The variation in metric measurements between replicate slides within stallions was small, indicating that replicate slide analysis probably is not necessary for clinically normal stallions. Coefficients of variation were generally less than 11% for metric measurements between stallions, and were less than 4% within stallions. This study revealed that a high degree of statistical power can be achieved when using these new, standardized specimen preparation and objective analysis techniques. Such power makes possible the detection of subtle differences between clinically normal stallions, and may facilitate accurate detection of abnormal fertility (ie, subfertility) in stallions.

Ultrasound-guided brain biopsy of the cingulate gyrus and the head of the caudate nucleus was performed in clinically normal dogs. Dogs survived the surgery, and neurologic deficits were not detected in the 14-day postoperative period. Magnetic resonance imaging detected changes in the brain associated with biopsy in 9 dogs (90%) immediately after surgery and in 6 dogs (60%) 14 days after surgery. Fourteen days after surgery, sonography of the brain, performed through the skin overlying the burr hole, detected changes associated with biopsy in 9 dogs (90%). Histopathologic changes evident in the brain 14 days after surgery consisted of focal malacia and hemorrhage with associated subacute encephalomeningitis. Postmortem examination indicated that the biopsy specimen was accurately obtained from the desired site in 9 dogs (90%). Tissue specimens suitable for histologic examination were obtained from 10 dogs (100%). Accuracy and low morbidity of ultrasound-guided biopsy indicate that this may be a useful technique for diagnosis of focal brain disease in dogs.

Chicken egg yolk IgG can be absorbed and transferred as efficiently as colostral antibodies in the blood of neonatal pigs. Egg yolk IgG has a half-life of 1.85 days in newborn pig serum. This is shorter than the reported half-life (12 to 14 days) of homologous IgG in serum of pigs. Similar to colostral antibodies, egg yolk IgG absorption from intestine ceased at about 34 hours of age, after a logarithmic decrease in absorption rate from birth. Egg yolk IgG absorption inhibition time in the gastrointestinal tract took 1.73 hours to decrease by half. Egg yolk IgG was protective against experimentally induced diarrhea in pigs when it was administered at high dose, and multiple dosing was instituted. Adverse effects were not observed when chicken egg yolk IgG was administered orally to pigs.