An efficient, single-step method for purification of the 110-kilodalton (kDa) hemolysin of Actinobacillus pleuropneumoniae was developed. An immunoaffinity column was made by cross-linking murine monoclonal antibody 8C2 to the 110-kDa hemolysin of A pleuropneumoniae strain J45 serotype 5 to protein A-agarose beads. Purified hemolysin with high hemolytic activity was obtained after washing the column with phosphate-buffered saline solution, and eluting the hemolysin with 50 mM diethylamine, pH 11.0. The same column was also used to purify the hemolysin from A pleuropneumoniae strain 4074 serotype 1. The purification procedure could be completed within 5 hours, and almost 50% of the total hemolytic activity and hemolysin protein was recovered in pure form.

Isoelectric focusing was performed on the soluble proteins of whole-body and excretory-secretory products (ESP) of Fasciola hepatica and Fascioloides magna. Adult F hepatica flukes were recovered from experimentally infected sheep and ESP obtained from the flukes; portions of liver were cut and frozen at -70 C. Fascioloides magna adults were collected from naturally infected white-tailed deer and ESP obtained; portions of liver were collected from noninfected white-tailed deer. Adult flukes and their host tissues were homogenized and centrifuged; protein concentrations with their ESP were determined and adjusted to < 2.50 mg/ml. Seven ESP samples from F hepatica and 1 from Fascioloides magna were subjected to isoelectric focusing with the 2 species of fluke and their respective host liver homogenates. After separation, gels were stained with silver and scanned on a laser densitometer. Protein banding patterns of the 2 species of flukes were dissimilar. In the pH range of 3.5 to 9.6, the body protein had approximately 30 peaks and ESP about 23 peaks in both species. Overall banding patterns of the body protein and ESP of both species were distinct from those of respective host tissues. Of the peaks reported as dominant, 3 of the body protein and 2 of ESP were shared between the 2 species. Fascioloides magna had more dominant peaks than F hepatica. This technique of soluble protein isoelectric focusing is simple and reproducible, and the 2 fluke species can easily be differentiated by this technique, as well as by morphologic characteristics.

Concentration of total proteins was measured and sodium dodecyl sulfate-polyacrylamide gel disk electrophoresis was performed on tear and plasma samples obtained from 26 healthy dogs, and the results were compared. Mean +/- SEM concentration of total proteins in tears was 0.63 +/- 0.04 g/dl, and significant effects of age or gender were not found. The protein composition of tears in dogs was complex, and bands from light and heavy chains of immunoglobulins were identified by electrophoresis.

Polypeptides from whole cell preparations of Pasteurella multocida serotypes A:12 and A:3 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Antigens were detected by immunoblot analysis, using sera from 3 groups of rabbits. Sera were obtained from rabbits inoculated intranasally with P multocida serotype A:12 or A:3, from rabbits maintained in a rabbitry with enzootic P multocida A:12 infection, and from rabbits maintained in a rabbitry with enzootic P multocida A:3 infection. Immunoblot analyses of pre- and postinoculation sera from experimentally infected rabbits, using serotype A:12 antigen, revealed 3 polypeptides with approximate molecular mass of 28, 30, and 37 kDa that consistently detected antibodies after P multocida-induced infection. Sera from rabbits naturally infected with either serotype, tested against serotype A:12 and A:3 antigens, detected the same polypeptides in both serotypes. Thus, immunologic reactivity to these polypeptides may be useful for serologic detection of P multocida infection.

In this study, we described water-insoluble proteins extracted from the germinative regions (stratum internum and coronary band epithelium) and the cornified outer surface (stratum medium) of the equine hoof wall. Two major types of polypeptides were identified: the intermediate filaments (IF) and the IF-associated proteins. The IF, including keratins, composed a major portion of this fraction, had electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the range of 40 to 80 kDa, and reacted with acidic or basic keratinspecific monoclonal antibodies. Differences in the composition of keratins between germinative layers and the stratum medium were seen. Another less well-characterized group of polypeptides associated with the IF also were extracted with the water-insoluble polypeptide fraction. These associated proteins had an apparent molecular weight between 10 and 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and contained a higher percentage of sulfur-containing amino acids than did the IF. Water-insoluble protein fractions compared favorably with those found in other less-specialized keratinizing tissue with respect to size, immunoreactivity with monoclonal antibody, and amino acid composition.

Protein concentration was determined, using the Bradford technique, in tears from cats with normal corneas and from cats with corneal sequestrum. Tears from the former group contained 5.81 +/- 2.29 mg of protein/ml; those from cornmeal sequestrum-affected cats contained 6.21 +/- 2.21 mg/ml. Difference between the 2 values was not significant. Molecular weight determination was made, using 4 to 20% sodium dodecyl sulfate-polyacrylamide gels. Molecular mass of proteins ranged from 263 to 14 kDa. There was no detectable difference in the band patterns for the 2 groups.

Four healthy calves were inoculated with Pasteurella haemolytica serotype 1 by instillation of a broth culture into the middle nasal meatus of the left nostril. Four weeks later, calves were exposed to infectious bovine rhinotracheitis virus by aerosol into both nostrils. All calves became ill, from approximately day 3 through day 10 after virus exposure, and shed increased amounts of nasal mucus. Two calves were induced to shed P haemolytica by the virus infection, and 2 calves required reinoculation with P haemolytica for nasal passages to become actively colonized. Elastase activity in nasal mucus increased about 15-fold within 3 days and peaked about 60-fold over baseline by 7 days after virus exposure. Activity of N-acetyl-beta-D-glucosaminidase, a measure of cell damage and serum leakage, increased slightly by day 3 and reached plateau on day 5, almost threefold over baseline activity. Protein and carbohydrate content increased at a rate similar to that of N-acetyl-beta-D-glucosaminidase activity with about 12-fold and sixfold increases, respectively. None of the variables returned to baseline by 19 days after virus exposure, Increased elastase activity preceded colonization by P haemolytica and decreasing elastase activity preceded decreasing P haemolytica concentration in the nasal secretions. A causal relation between elastase activity and P haemolytica colonization could be mediated by cleavage of epithelial cell surface fibronectin and exposure of receptors.

Concentrations of serum and vitreous humor constituents at time of death, and concentrations of vitreous humor constituents at time of death and at 7 postmortem intervals were compared in 70 domestic, female New Zealand White rabbits (Oryctolagus cuniculus). Urea nitrogen concentration was significantly (P = 0.0094) different, but was linearly correlated in serum and vitreous humor at time of death and at the 4- and 8-hour postmortem intervals. Concentrations of gamma-glutamyltransferase were not significantly different in serum and vitreous humor at time of death, nor were concentrations significantly different in vitreous humor at time of death and at the 4-hour postmortem interval. The vitreous humor concentrations of glucose, triglycerides, sodium, potassium, cholesterol, total protein, albumin, lactate dehydrogenase, creatine kinase, aspartate transaminase, bilirubin, cortisol, and IgG were neither similar to nor predictive of serum constituents. Vitreous humor can be used as a source for estimates of serum urea nitrogen and gamma-glutamyltransferase up to 8 and 4 hours after death, respectively.

Monensin is an ionophoretic antibiotic, which selectively transports alkali metal cations across biological membranes. In growing swine, monensin toxicosis causes acute, degenerative cardiac and skeletal myopathy resembling vitamin E-selenium deficiency. Selenium is an essential trace element incorporated in glutathione peroxidase (GSH-Px), an antioxidant enzyme system that protects subcellular membranes. In our study, we examined the effects of monensin on body weight, Se balance, antioxidant status, and serum concentrations of selected minerals in growing pigs that were genetically hypo- or hyperselenemic (hypo-Se and hyper-Se, respectively). Three groups of eight 8-week-old pigs, each comprised of 4 hypo-Se and 4 hyper-Se pigs (76.4 +/- 3.0 and 106.3 +/- 10.3 ng of Se/ml of serum, respectively), were fed standard diets containing 0.1 mg of supplemental Se/kg of body weight, and either 0, 200, or 400 mg of monensin/kg for a 77-day period, followed by a 28-day monensin withdrawal period. On days 0, 7, 28, 56, 70, and 98, all pigs were weighed and blood was collected for determination of serum GSH-Px, creatine phosphokinase, and aspartate transaminase values, as well as serum concentrations of vitamin E, Se, Ca, Cu, Fe, K, Mg, Na, P, and Zn. Significance of main effects of monensin treatment, genetic Se status, and their interactions was tested by Fisher's variance ratio test, followed by conditional comparison of treatment means with a Bonferroni test. Signs of monensin toxicosis were not observed and monensin consumption had no effect on body weight, or serum creatine phosphokinase, aspartate transaminase, or Se values. However, pigs consuming monensin had consistently higher serum GSH-Px activities, possibly because of increased synthesis of this adaptive antioxidant enzyme. Interactions were not found between monensin and genetic Se status. Hyperselenemic pigs were heavier and had higher serum Se and GSH-Px values than hypo-Se pigs. Furthermore, hypo-Se and hyper-Se pigs were hypo- and hypercupremic, respectively, suggesting genetic regulation of copper status. It is likely that pigs with inadequate antioxidant status (hyposelenemia, hypocupremia) are more susceptible to diseases associated with cellular membrane damage, such as vitamin E-Se deficiency disease and monensin toxicosis.