Comparison of the efficacy of a subunit and a live streptomycin-dependent porcine pleuropneumonia vaccine.

OBJECTIVE: To evaluate the efficacy of two new-generation porcine pleuropneumonia vaccines when challenged with Australian isolates of Actinobacillus pleuropneumoniae of serovars 1 and 15. DESIGN: The Porcilis APP vaccine and an experimental streptomycin-dependent strain of A pleuropneumoniae were evaluated in a standardised pen trial. Each vaccine/challenge group consisted of 10 pigs. RESULTS: With the serovar 1 challenge, the Porcilis APP vaccine and the live vaccine, compared with the control group, gave significant protection in terms of clinical signs, lung lesions, re-isolation scores and average daily gain (ADG) postchallenge. Only the Porcilis APP vaccine provided significant protection against mortality. In the serovar 15 challenged pigs, the only significant difference detected was that the Porcilis APP vaccinated pigs had a better postchallenge ADG than the controls. None of the Porcilis APP vaccinated pigs showed signs of depression postvaccination and none were euthanased after challenge with either serovar 1 or 15. The pigs vaccinated with the live vaccine showed obvious depression after each vaccination and a total of 3 pigs were euthanased after challenge (one with serovar 1 and two with serovar 15). CONCLUSIONS: Both of the vaccines provided significant protection against a severe challenge with serovar 1 A pleuropneumoniae. Neither vaccine was effective against a serovar 15 A pleuropneumoniae challenge. There was evidence that the Porcilis APP vaccine did provide some protection against the serovar 15 challenge because the ADG, after challenge of pigs given this vaccine, was greater than the control pigs.

An evaluation of the role of antibodies to Actinobacillus pleuropneumoniae serovar 1 and 15 in the protection provided by sub-unit and live streptomycin-dependent pleuropneumonia vaccines.

OBJECTIVE: To evaluate the serological response of pigs receiving either the Porcilis APP vaccine or a modified live vaccine based on a streptomycin-dependent (SD) strain of Actinobacillus pleuropneumoniae, and then challenged with an Australian isolate of A. pleuropneumoniae of either serovar 1 or 15 as a means of understanding the protection provided by both vaccines against serovar 1 but not against serovar 15. DESIGN: The serological tests evaluated were serovar-specific polysaccharide ELISA tests (for serovar 1 and 15), ELISA tests for antibodies to three A. pleuropneumoniae toxins (ApxI, ApxII and ApxIII) as well as to a 42 kDa outer membrane protein (OMP), a haemolysin neutralisation (HN) assay and immunoblotting. The tests were used to detect antibodies in vaccinated pigs that had been shown to be protected against serovar 1 but not serovar 15. RESULTS: In the polysaccharide antigen ELISA assays, both vaccines resulted in a significant rise in the titre in the serovar 1 ELISA but not the serovar 15 ELISA. The Porcilis APP vaccinated pigs showed a significant response in the ApxI, ApxIII and 42 kDa OMP ELISA. In the ApxII ELISA, all pigs tested (the Porcilis APP vaccinates and the controls) were positive on entry to the trial. In the HN assay, the Porcilis APP vaccinated pigs showed a significant response after one dose while the SD vaccinated pigs required two doses of vaccine before a marked rise in titre was induced. Immunoblotting revealed that neither vaccine generated antibodies that recognised the ApxIII produced by serovar 15. CONCLUSIONS: The failure of these vaccines to provide protection against serovar 15 may be due to novel virulence factors possessed by serovar 15, significant differences between the ApxIII toxin of serovar 15 and those present in the Porcilis APP vaccine or failure by both vaccines to induce antibodies to the serovar 15 specific polysaccharide.

Normal anatomical and histochemical characteristics of the lacrimal glands in the American bison and cattle.

Dorsal lacrimal glands, superior glands of the third eyelid and Harderian glands (deep gland of the third eyelid) from 19 bison and 18 cattle free of apparent ocular disease were examined to compare the normal anatomical properties of these glands. All glands were characterized and measured (length and width). The gross anatomy of the dorsal lacrimal glands was similar, with the exception of a bipartite gland in cattle. The bison's superior gland of the third eyelid and Harderian gland was longer as compared with cattle. A subset of the bison and cattle samples (five bison and five cattle) was sectioned for histological and histochemical analysis. The histology of the dorsal lacrimal and superior gland of the third eyelid revealed tubuloalveolar cells with basophilic vacuolated cytoplasm in bison and eosinophilic granular cytoplasm in cattle. The Harderian glands consisted of a tubuloalveolar anterior part combined with large lumens acini lined with cuboidal epithelium in the posterior part; the posterior part of the bison Harderian gland was more predominant than in cattle samples. Mucosubstance histochemistry revealed acidic and neutral glycoproteins with similar staining patterns in all glands of both species.

Circulating cortisol, tumor necrosis factor-alpha interleukin-1beta, and interferon-gamma in pigs infected with Actinobacillus pleuropneumoniae.

This study evaluated the time course of systemic cytokine concentrations in an acute model of pneumonia in pigs challenged intranasally with Actinobacillus pleuropneumoniae. Feed intake and serum cortisol were measured as overt clinical and systemic markers of disease onset, respectively, and serum tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma as representative systemic inflammatory markers. Crossbred barrows (n = 15), approximately 5 wk of age, were used in the study. Pigs were housed in an environmentally controlled facility at 25 degrees C and under continuous illumination in pens measuring approximately 1.5 m2. Pigs had free access to water and an unmedicated diet. Approximately 1 wk prior to disease challenge, pigs were fitted nonsurgically with venous catheters. At challenge, pigs were given 5 x 10(8) CFU Actinobacillus pleuropneumoniae intranasally (n = 8) or a similar volume of sterile growth media intranasally (Control; n = 7). Feed intake was estimated by the change in feeder weight at 12-h intervals from -12 to 72 h relative to the time of disease challenge. Blood sampling began 12 h prior to challenge and continued until 72 h after challenge. Pigs were sampled at -12, -6, and 0 h, then at 90-min intervals until 12-h post-challenge, continuing at 3-h intervals until 24-h post-challenge, then again at 6-h intervals until 72 h after challenge. Serum was harvested and frozen until assayed for cortisol, tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma. Feed intake was reduced in Actinobacillus pleuropneumoniae pigs during the intervals 0 to 12 h (P < 0.001), 24 to 36 h (P < 0.001), 48 to 60 h (P <0.05), and 60 to 72 h (P < 0.05). TheActnobacillus pleuropneumoniae-challenged pigs had elevated serum cortisol from 180-min to 18-h post-challenge (P < 0.001) and also at 36 (P < 0.05), 42 (P < 0.001), and 60 (P < 0.05) h following infection. Circulating cytokines were not affected by disease challenge. Thus, in this experimental model of pneumonia, weaned pigs demonstrated expected behavioral and endocrine characteristics of disease in the absence of significant changes in circulating inflammatory cytokines.

Biochemical alteration of membrane-associated IL-6 RI (80-kDa) in adherent macrophages and vascular endothelium.

The potential biochemical mechanisms that mediate the 'shedding' of soluble IL-6 RI (80-kDa) receptor fragments in populations of adherent macrophages were explored. Stimulated macrophages displayed proportional increases in both the expression of membrane-associated IL-6 RI (80-kDa) and the release of soluble receptor fragments. The use of protease inhibitors implicated thiol/cysteine and carboxyl/aspartate proteases in this process. Cathepsin-D depleted membrane-associated IL-6 RI (80-kDa) complexes and generated soluble receptor fragments. A carboxyl/aspartate protease from activated macrophages isolated utilizing pepstatin-A affinity chromatography, was also found to affect membrane-associated IL-6 RI (80-kDa) complexes and generate soluble receptor fragments. Most likely, this fraction corresponded to cathepsin-D based upon its origin, pepstatin-A binding avidity, Hb-PAGE zymography, and hydrolysis of an enzyme-specific substrate. We conclude that cathepsin-D can generate soluble fragments of IL-6 RI (80-kDa) expressed by both macrophages and vascular endothelium.

Biochemical mechanisms that interact with membrane-associated IL-1 RII (60-kDa decoy) receptors in populations of adherent macrophages and vascular endothelium.

The aim of this investigation was to identify the potential biochemical mechanisms that alter the integrity of membrane-associated IL-1 RII (decoy) receptor complexes expressed by populations of adherent macrophages and vascular endothelium. The initial research strategy utilized to achieve this objective involved delineating the ability of macrophage activation or exposure of macrophages and vascular endothelium to a spectrum of enzyme proteases to influence the expression of membrane-associated IL-1 RII (decoy) or generate soluble fragments of this receptor complex. Results from these investigations revealed that stimulated macrophages displayed proportional increases in both the expression of membrane-associated IL-1 RII (decoy) and release of soluble receptor fragments. Exposure of macrophages and vascular endothelium to the reference proteases discovered the ability of cathepsin-D to biochemically deplete membrane-associated IL-1 RII (decoy) in addition to generating soluble fragments of this receptor complex. Complementary investigations isolated a carboxyl/aspartate protease from activated macrophages utilizing pepstatin-A affinity chromatography. Exposure of vascular endothelium to pepstatin-A binding proteins resulted in a detectable depletion of membrane-associated IL-1 RII (decoy) and generation of soluble receptor fragments. Evaluation of pepstatin-A binding proteins by SDS-PAGE identified a primary protein fraction with a molecular mass of 47-52 kDa that closely correlates with the known molecular size of leukocyte cathepsin-D fractions. Macrophage pepstatin-A binding protein fractions evaluated by nondenaturing haemoglobin-substrate PAGE (Hb-PAGE) analysis detected a lucent proteolytic band at 47-52 kDa. Macrophage pepstatin-A binding proteins also hydrolyzed a synthetic enzyme-specific substrate that selectively recognizes cathepsin-D biochemical activity. In conclusion, the leukocyte carboxyl/aspartate protease cathepsin-D can biochemically alter the integrity and generate soluble fragments of membrane-associated IL-1 RII (60-kDa decoy) receptor complexes expressed by macrophages and vascular endothelium.

Synthesis and evaluation of novel aldose reductase inhibitors: Effects on lens protein kinase Cgamma.

PURPOSE: To synthesize novel aldose reductase inhibitors (ARI) that will normalize losses in protein kinase Cgamma (PKCgamma) observed during diabetes and galactosemia. METHODS: ARI were synthesized as tricyclic pyrones 1-6 (HAR-1 through HAR-6) from 3-methyl-1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b][1]benzopyran and (5aS,7S)-7-isopropenyl-3-methyl-1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b][1]benzopyran and were tested by inhibition of aldose reductase enzyme activity in vitro and by inhibition of polyol formation in lens epithelial cells in culture. Identified compounds were further tested in galactosemic rat lens in vivo for (a) normalized PKCgamma levels by Western blot, (b) reduction of phosphorylation of the gap junction protein Cx46 by analyses of co-immunoprecipitated proteins, and (c) by normalization of gap junction activity as measured by dye transfer. RESULTS: HAR-1 (1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b][1]benzopyran-3-acetic acid) was identified as an ARI with IC50 for aldose reductase inhibition at 2 nM. Polyol accumulation in lens epithelial cells was reduced by 80% at 10 microM. Rats fed 40% galactose for 9 days had an 80% reduction in PKCgamma levels which were normalized by HAR-1 at 100 mg/kg/day, fed orally. Phosphorylation of Cx46 was increased by 50% and this was normalized in HAR-1 treated rats (6 day treatment). Gap junction activity of galactosemic rats was reduced by 55% and this was normalized by HAR-1 in six day-treated rats. CONCLUSIONS: HAR-1 is a novel ARI which normalized losses of PKCgamma, changes in Cx46 phosphorylation, and gap junction activity.

Relationship between mean body surface temperature measured by use of infrared thermography and ambient temperature in clinically normal pigs and pigs inoculated with Actinobacillus pleuropneumoniae.

OBJECTIVE: To determine the relationship between ambient temperature and mean body surface temperature (MBST) measured by use of infrared thermography (IRT) and to evaluate the ability of IRT to detect febrile responses in pigs following inoculation with Actinobacillus pleuropneumoniae. ANIMALS: 28 crossbred barrows. PROCEDURES: Pigs (n = 4) were subjected to ambient temperatures ranging from 10 to 32 C in an environmental chamber. Infrared thermographs were obtained, and regression analysis was used to determine the relationship between ambient temperature and MBST. The remaining pigs were assigned to groups in an unbalanced randomized complete block design (6 A pleuropneumoniae-inoculated febrile pigs [increase in rectal temperature > or = 1.67 C], 6 A pleuropneumoniae-inoculated nonfebrile pigs [increase in rectal temperature < 1.67 C], and 12 noninoculated pigs). Infrared thermographs and rectal temperatures were obtained for the period from 2 hours before to 18 hours after inoculation, and results were analyzed by use of repeated-measures ANOVA. RESULTS: A significant linear relationship was observed between ambient temperature and MBST (slope, 0.40 C). For inoculated febrile pigs, a treatment X method interaction was evident for rectal temperature and MBST, whereas inoculated nonfebrile pigs only had increased rectal temperatures, compared with noninoculated pigs. A method X time interaction resulted from the longer interval after inoculation until detection of an increase in MBST by use of IRT. CONCLUSIONS AND CLINICAL RELEVANCE: Infrared thermography can be adjusted to account for ambient temperature and used to detect changes in MBST and radiant heat production attributable to a febrile response in pigs.

Serologic detection of Actinobacillus pleuropneumoniae in swine by capsular polysaccharide-biotin-streptavidin enzyme-linked immunosorbent assay.

Serologic detection of Actinobacillus pleuropneumoniae infections in swine have been problematic due to antigenic cross-reactivity of Apx toxins, lipopolysaccharide, and outer membrane proteins between A. pleuropneumoniae serotypes and other bacterial species. To maximize serologic specificity and sensitivity, we developed an assay that uses highly purified A. pleuropneumoniae capsular polysaccharide (CP) conjugated to biotin, which is then bound to streptavidin-coated enzyme-linked immunosorbent assay (CP-BS-ELISA) plates. This assay was used to test a panel of 240 serum samples from pigs prior to challenge, after challenge with bacterial species other than A. pleuropneumoniae, or after challenge with A. pleuropneumoniae serotype 1, 5, or 7. Overall assay results for the individual sera tested were reproducible on the same day and on separate days. The sensitivity of the assay was 100% by ELISAs with biotin-CPs of serotypes 1 and 7 and 87.5% by ELISAs with biotin-CP of serotype 5. Specificity was 100% by ELISAs with biotin-CPs of serotypes 1 and 5 and 94.5% by ELISAs with biotin-CP of serotype 7. The biotin-CPs of at least three A. pleuropneumoniae serotypes could be combined for use in a screening assay to detect antibodies to CPs from strains of different serotypes. In conclusion, the CP-BS-ELISA proved to be a serotype-specific and species-specific assay with high sensitivity for the identification of pigs exposed to A. pleuropneumoniae.

Cloning and immunologic characterization of a truncated Bordetella bronchiseptica filamentous hemagglutinin fusion protein.

Filamentous hemagglutinin (FHA) is an outer-membrane associated adhesin conserved within the genus Bordetella. FHA provides protection against B. pertussis infections in humans and is a component of acellular whooping cough vaccines. Furthermore, FHA serves as a protective antigen in several animal models of infection with B. bronchiseptica and may serve as a protective antigen of canine bordetellosis. In this study, polyclonal anti-B. pertussis FHA antiserum was used to identify an immunoreactive clone from the genomic DNA library of a canine B. bronchiseptica field isolate. The nucleotide and predicted amino acid sequences of the immunoreactive clone were compared to fhaB and FhaB from B. pertussis revealing 94% identity at the nucleic acid level, and 86% identity at the protein level. A truncated fusion protein (FHAt) was prepared which included a conserved domain homologous to the immunodominant region in the FHA of B. pertussis [Leininger E, Bowen S, Renauld-Mongen G, Rouse JH, Menozzi FD, Locht C, Heron I, Brennan MJ. Immunodominant domain present on the Bordetella pertussis vaccine component filamentous hemagglutinin. J. Infect. Dis. 1997;175:1423-1431; Wilson DR, Siebers A, Finlay BB. Antigenic analysis of Bordetella pertussis filamentous hemagglutinin with phage display libraries and rabbit anti-filamentous hemagglutinin polyclonal antibodies. Infect. Immun. 1998;66:4884-4894]. FHAt was shown to be safe and antigenic in rabbits. FHAt induced the formation of antibodies that inhibit the hemagglutination associated with full length B. pertussis FHA, and inhibit adherence of B. bronchisepitca to canine fibroblasts by as much as 65%. This information may have implications for the development of safe and efficacious subunit vaccines for the prevention of canine bordetellosis and may contribute to future acellular whooping cough vaccines.

Pasteurellaceae isolated from tonsillar samples of commercially-reared American bison (Bison bison).

As commercial producers of American bison (Bison bison) become more numerous, concerns relative to bison health management increase. Since loss due to respiratory disease associated with Pasteurella and related Pasteurellaceae is a major concern for cattle producers, a study was conducted to determine what types of Pasteurellaceae are carried by bison to evaluate the potential of pneumonic pasteurellosis in bison herds where management practices are comparable to those used for cattle. Tonsillar biopsies, collected in May (n = 29) and August (n = 25) 1997 from 24- to 30-month-old bison bulls, at the time of slaughter were cultured for Pasteurellaceae. Pasteurella spp. were isolated from all the samples collected in May. These included isolates identified as P. haemolytica, trehalosi, testudinis, and multocida subsp. multocida a and multocida b. Actinobacillus spp. and Haemophilus somnus were also isolated from some samples. Pasteurella spp., haemolytica, trehalosi, and multocida subsp. multocida a, multocida b and septica, plus 2 nonspeciated indole-positive biotypes, U2 and U16, were isolated from the second group of tonsil samples. Most of these organisms, including P. haemolytica, P. multocida subsp., and H. somnus are associated with disease in domestic livestock and should be regarded as potential pathogens for bison, particularly in animals which become stressed by management practices commonly used with cattle such as herding, crowding, and shipping.

Strain- and growth condition-dependent variability in outer membrane protein expression by Bordetella bronchiseptica isolates from dogs.

OBJECTIVE: To characterize strain-dependent and growth condition-dependent variability in outer membrane protein (OMP) expression of Bordetella bronchiseptica isolates from dogs and evaluate the systemic immune response to OMP of B bronchiseptica among infected dogs. SAMPLE POPULATION: 8 strains of B bronchiseptica isolated from dogs, including a historic reference strain, 2 commercially available vaccine strains, and 5 field strains, and serum samples collected from 3 specific-pathogen-free (SPF) dogs before and 1 month after infection with B bronchiseptica. PROCEDURE: OMP were isolated from cultures in the late exponential phase of growth and compared among strains and, within strains, among growth conditions by means of polyacrylamide gel electrophoresis and immunoblotting. Serum samples were probed with OMP from 1 of the field strains. RESULTS: Strain-dependent variability in OMP profiles and growth condition-dependent and strain-dependent variability in expression of filamentous hemagglutinin (FHA) and pertactin was found, along with heterogeneity of the pertactin proteins produced by these B bronchiseptica strains. All 3 SPF dogs seroconverted to proteins with estimated molecular masses of 200 and 66 kDa, suggesting that FHA and pertactin were involved in the immunologic response of these dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that there is growth condition and strain variability in expression of OMP, FHA, and pertactin proteins produced by B bronchiseptica. This information could be useful in the improvement of vaccines for prevention of bordetellosis in dogs.

Evaluation of canine Bordetella bronchiseptica isolates using randomly amplified polymorphic DNA fingerprinting and ribotyping.

Bordetella bronchiseptica is a respiratory tract pathogen in a variety of species. Previous studies suggest little genetic variation among canine B. bronchiseptica isolates. The degree of genetic diversity in 26 canine B. bronchiseptica strains was evaluated using randomly amplified polymorphic DNA (RAPD) fingerprinting and ribotyping. Strains evaluated include historic reference strains (N=3). vaccine strains (N=5) and clinical isolates (N=18). RAPD fingerprinting with the 10-nucleotide primer OPA-4 resulted in four distinct fingerprint patterns. RAPD fingerprinting consistently separated four previously characterized electromorphotype (EMT) 6 strains into two fingerprint types. Ribotyping, using the restriction endonuclease PvuI, resulted in six distinct ribotypes. With the exception of vaccine strains, considerable genetic diversity exists in the canine B. bronchiseptica isolates examined. These findings indicate the genetic variability within canine strains of B. bronchiseptica is greater than appreciated previously. Additionally, OPA-4 RAPD fingerprinting and PvuI ribotyping will be useful tools in epidemiologic studies of canine B. bronchiseptica isolates.

Actin polymerization enhances Pasteurella haemolytica leukotoxicity.

Pasteurella haemolytica leukotoxin is cytotoxic to bovine leukocytes, causing increased cell membrane permeability, osmotic swelling, release of cytosolic proteins and cell lysis. These studies were designed to test if leukotoxin causes release of the cytoskeletal protein, actin, from bovine leukemia cells and if purified actin-influenced bacterial growth or leukotoxin production. Culture supernatants caused a 7-fold decrease in viability of bovine leukemia cells and increased cell permeability that was accompanied by release of beta-actin into the cell culture supernatant. Exposing P. haemolytica to purified actin solutions induced the conversion of monomeric G-actin to polymerized F-actin. This conversion was partially inhibited by bovine P. haemolytica immune, but not pre-immune, serum. Loss of streptomycin resistance following treatment of the organism with acridine orange ablated the polymerizing activity. Incubation of P. haemolytica in the presence of purified F-actin did not affect growth but resulted in culture supernatant that had 3.0-3.9-fold greater leukotoxicity compared to medium alone or medium containing G-actin, heat-denatured actin or albumin. The effect of actin on leukotoxicity was concentration-dependent and directly associated with increases in secreted leukotoxin. The interaction between P. haemolytica and actin is potentially detrimental to the host by inducing polymerization of actin into insoluble filaments and by enhancing leukotoxicity.

A mechanism of TNFR type II (75 kDa) "shedding" in macrophages.

Adherent macrophage populations derived from monocytes isolated from peripheral blood were evaluated for their ability to "shed" the membrane-associated receptor for TNF-alpha (TNFR) following exposure to a calcium ionophor (A23187) and a synthetic chemotactic peptide (fMLP) reagent. A soluble fraction of TNFR was detected in "cell-free" supernatant produced by stimulated macrophage populations applying 125I-TNF-alpha and biotinylated TNF-alpha ligand-binding analysis (96-well format) in combination with conventional autoradiographic techniques. Approximate molecular weight of the shed TNFR glycoprotein fraction was estimated to be 75 kDa based on interpretation of nondenaturing PAGE gels transferred laterally onto sheets of nitrocellulose membrane subsequently probed by ligand-binding analysis applying 125I-TNF-alpha and biotinylated TNF-alpha as detection modalities. Immunorecognition techniques were also employed to detect TNFR fragments shed from macrophages using biotinylated anti-TNFR Type II (75 kDa) monoclonal antibody in combination with conjugated strepavidin:HRPO and a chemiluminescent substrate reagent. In an effort to identify the class of enzyme directly mediating TNFR Type II (75 kDa) shedding, a spectrum of carboxyl- (e.g., aspartate), hydroxyl- (e.g., serine), thiol (e.g., cysteine), and metalo- (e.g., Ca2+, Mg2+) protease-inhibiting agents were evaluated. Experimental findings implied that a carboxy (aspartate) peptidase, and possibly to a lesser extent, serine (hydroxyl), and thiol (cysteine) peptidases participate in macrophage TNFR Type II (75 kDa) shedding phenomena. Subsequent investigations demonstrated that the carboxy (aspartate) peptidase cathepsin-D promoted liberation of TNFR Type II (75 kDa) in unactivated populations of adherent macrophages. In an effort to complement these observations, a protein fraction with presumed carboxy (aspartate) protease activity was isolated from the cell-free supernatant generated by activated populations of adherent macrophages using immobilized pepstatin-A beaded agarose. Exposure of unstimulated populations of adherent macrophages to the partially purified pepstatin-A binding protein fractions resulted in the liberation of a soluble TNFR Type II (75 kDa) fragment based on interpretation of ligand-binding and immunorecognition analysis of samples developed by SDS-PAGE/PAGE format and transferred onto sheets of nitrocellulose membrane. The molecular weight of the macrophage pepstatin-A binding protein fraction was estimated to be 47-52 kDa with lesser bands also visible at approximately 26-32 kDa, and 100 kDa based on SDS-PAGE analysis. Nondenaturing hemoglobin-PAGE substrate gel analysis of protein fractions possessing pepstatin-A binding-avidity detected a protease with a molecular weight of approximately 47-52 kDa that proteolytically digested hemoglobin, in addition to a synthetic cathepsin-D specific peptide substrate. Collective interpretation of these experimental findings directly corresponds with many of the physical (molecular) and functional (biochemical) characteristics known to be associated with the leukocyte carboxy (aspartate) peptidase cathepsin-D, which is a non-metaloprotease known to exert relatively limited proteolytic activity.

Actin enhances the haemolytic activity of Escherichia coli.

Actin is a major cytoskeletal protein of mammalian muscle and non-muscle cells. Exposure of cells to soluble factors that damage cell membranes results in the release of actin into the extracellular spaces. The alpha-haemolysin (HlyA) of Escherichia coli is the prototype RTX (repeat in toxin) toxin and is thought to be important in virulence because of its ability to lyse cells by formation of pores in the cell membrane. These studies were conducted to determine if actin influences growth and haemolytic activity of E. coli. Growth of E. coli in the presence of actin resulted in culture supernatant haemolytic activity that was 2.4-, 2.7- and 3.3-fold greater than that of E. coli grown in medium containing BSA, non-supplemented medium, or medium containing heat-denatured actin, respectively. The enhanced haemolytic activity occurred only when actin was present during the growth phase and there was no effect when actin was added to culture supernatants containing haemolysin. The increased haemolytic activity by actin was concentration-dependent, detectable in early-exponential-phase growth, and associated with increased concentrations of secreted HlyA by Western blotting. Actin induced a 2.9-fold increase in alkaline phosphatase activity in E. coli CC118 with a TnphoA insertion in the hlyB determinant of the recombinant haemolysin plasmid pWAM04. These results indicate that extracellular actin enhances haemolysin production by E. coli and may have implications in the pathogenesis of E. coli infections.

Infectious bovine keratoconjunctivitis: a review.

The economic impact of infectious bovine keratoconjunctivitis (IBK) warrants continued investigation of the mechanisms by which Moraxella bovis survives on and colonizes the corneal surface. Virulent strains of M bovis produce hemolysin and exhibit different plasmid profiles than nonvirulent strains. Interactions among host, environment, vector, season, and concurrent infection influence the prevalence of IBK. Mycoplasma sp. or infectious bovine rhinotracheitis virus may enhance or hasten the disease process. The manifestations of IBK may range from mild conjunctivitis to severe ulceration, corneal perforation, and blindness. Treatment of IBK is dictated by economic considerations, intended animal use, and feasibility of administration. Antibiotic therapy is aimed at achieving drug concentrations in tears to meet or exceed the minimum inhibitory concentration for prolonged periods. At present, IBK is not a preventable disease. Affected animals must be separated from the herd and vector control vigorously instituted. Carrier animals must be identified and removed from the herd. Vaccination trials have been unsuccessful because of pili antigen cross-reactivity, variable strains, and uncontrolled environmental factors. Recent investigations have determined that M bovis may utilize host iron sources via iron-repressible outer membrane proteins and siderophores for growth. Elucidation of normal defense mechanisms of the bovine eye may lead to new strategies to enhance the immune response against M bovis.

Reduction of acyloxyacyl hydrolase activity in circulating neutrophils from cows after parturition.

Bovine neutrophils contain the enzyme acyloxyacyl hydrolase, which hydrolyzes the acyloxyacyl linkage of the two nonhydroxylated fatty acyl chains to two 3-hydroxy fatty acids in the highly conserved lipid A part of endotoxins with high specificity. This hydrolysis decreases the toxicity of lipid A, but the immunostimulatory capacity of endotoxins is largely maintained. In two trials, we studied the activity of acyloxyacyl hydrolase in neutrophils that had been isolated from the blood of 18 dairy cows around parturition. Between 10 and 26 d after parturition, the activity of acyloxyacyl hydrolase in neutrophils decreased approximately 20% below prepartum activity. At about 2 mo after parturition, acyloxyacyl hydrolase activity returned to prepartum values. Changes in acyloxyacyl hydrolase activity could not be attributed to changes in binding of lipopolysaccharides by the CD14 molecules on neutrophils or monocytes. We hypothesize that decreased acyloxyacyl hydrolase activity in neutrophils shortly after parturition is a factor that increases the susceptibility of dairy cows to coliform mastitis during early lactation.

Clinical and clinicopathologic abnormalities in greyhounds with cutaneous and renal glomerular vasculopathy: 18 cases (1992-1994)

OBJECTIVE: To determine clinical signs and clinicopathologic abnormalities in Greyhounds with cutaneous and renal glomerular vasculopathy and to determine whether there were any differences between dogs with and without renal azotemia. DESIGN: Retrospective study. ANIMALS: 18 Greyhounds. PROCEDURE: Results of CBC, serum biochemical analyses, urinalyses, coagulation tests, tests of RBC morphology, bacterial culture of blood samples, and serologic tests for Rickettsia rickettsii, Ehrlichia canis, E platys, and Leptospira interrogans were reviewed. Glomerular filtration rates and urine protein:creatinine ratios were determined in most dogs. t-Tests and a test of equality of proportions were used to compare dogs that developed renal azotemia with dogs that did not. RESULTS: None of the dogs was bacteremic or had serologic evidence of infectious disease. Ten dogs had renal azotemia, 16 had anemia, 11 had hypoalbuminemia, and 18 developed thrombocytopenia. Compared with dogs without renal azotemia, dogs with renal azotemia had significantly lower mean platelet count, hematocrit, and serum albumin concentration and significantly higher mean neutrophil count and creatine kinase activity. All 10 dogs with renal azotemia died or were euthanatized; 7 of 8 dogs without azotemia survived. CLINICAL IMPLICATIONS: Greyhounds with cutaneous and renal glomerular vasculopathy that developed renal azotemia had evidence of more severe systemic disease than did dogs that did not have azotemia and, despite supportive treatment, had a poorer prognosis.