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.

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.

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.

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.

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.

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.

Cytotoxic activity of doxorubicin "loaded" neutrophils against human mammary carcinoma (HTB-19).

Neutrophils were intra-cellularly "loaded" with the chemotherapeutic agent, doxorubicin applying a variety of incubation conditions in order to identify parameters which maximize chemotherapeutic incorporation, while simultaneously preserving optimal viability and chemotactic responsiveness. Doxorubicin "loaded" neutrophils (DLN) were produced in triplicate at different combinations of incubation conditions such as temperature (4 degrees C, 37 degrees C); duration (0, 1, 2 hours); and doxorubicin concentration (20, 40, 60 micrograms/ml). Chemotactic responsiveness of rinsed DLN preparations was subsequently assessed against the neutrophil peptide chemotactic agent, formyl methionyl leucyl phenylalanine (fMLP, 10(-6) M) utilizing a modified 96-well Boyden chemotactic chamber apparatus. Viable, fMLP-responsive DLN preparations were subsequently detected with MTT vitality staining reagent. At sub-physiological incubation temperatures (4 degrees C), profound declines in the viability of DLN preparations were detected when simultaneously incubated with doxorubicin formulated at concentrations greater than 10 micrograms/ml. In contrast, DLN preparations incubated at 37 degrees C displayed diminished viability only when incubated with doxorubicin formulated at a concentration of 60 micrograms/ml. Viable DLN populations were subsequently evaluated to determine their ability to exert in vitro cytotoxic activity against monolayer populations of human mammary carcinoma (HTB-19) propagated in a tissue culture environment. The lethal effect which DLN preparations inflicted towards HTB-19 populations was substantially greater than was observed with an equivalent population of untreated neutrophils. Maximal in vitro cytotoxic activity was detected with DLN preparations produced at 37 degrees C in the presence of doxorubicin formulated at a concentration of 40 micrograms/ml. In contrast, DLN preparations produced at an incubation temperature of 37 degrees C, and a doxorubicin concentration of 20 micrograms/ml displayed relatively lower levels of in vitro cytotoxic activity against HTB-19 monolayer populations. The degree of in vitro cytotoxic activity exerted against HTB-19 monolayer populations by DLN preparations was directly influenced by the duration of the challenge period. Maximal in vitro cytotoxic activity was observed when HTB-19 monolayer populations were challenged with DLN preparations for a period of 96-hours duration at 37 degrees C. Challenge periods of 48-hours duration produced levels of in vitro cytotoxic activity which were substantially lower than those observed for challenge periods of 96-hours duration. Optimal in vitro cytotoxic activity was recognized when DLN preparations were allowed to establish direct contact with HTB-19 monolayer populations at an estimated DLN:HTB-19 cellular ratio of approximately 5:1 (37 degrees C, CO2, 6%). Significantly less in vitro cytotoxic activity was recognized when DLN preparations were only permitted indirect cellular contact with HTB-19 monolayer populations which was achieved through the application of a semi-permeable 3 microM pore membrane partition. In vitro cytotoxic activity of DLN populations was not inhibited by the anti-oxidant agent, dimethyl sulfoxide (DMSO), but was inhibited in the presence of glutathione (GSH), superoxide dismutase (SOD), and vitamin E (alpha-tocopherol). Similarly, in vitro cytotoxic activity of DLN populations was also inhibited in the presence of sodium heparin (serine esterase inhibitor), and dexamethasone (inhibitor of neutrophil activation-degranulation phenomenon). Experimental results observed in these investigations collectively imply that the in vitro cytotoxic activity exerted by DLN preparations against HTB-19 populations is in part attributable to neutrophil-mediated cytotoxic immunity. This innate property of neutrophil populations involves their capacity to generate highly reactive oxygen "free" radical species (O2, HO, H2O2), and synthes

Identification of lactoferrin in bovine tears.

OBJECTIVE: To determine whether bovine tear film contains the iron-binding glycoprotein, lactoferrin. ANIMALS: 40 Adult Hereford, Angus, and Simmental cattle. PROCEDURE: Protein analysis: pooled bovine tears were used for protein analysis (size exclusion high-performance liquid chromatography [HPLC] fractionation). HPLC was used for tear analysis. A diode array detector was used (215 and 280 microns) for chromatogram analysis and comparisons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): protein electrophoresis was performed, using 7.5% running gels with 4% stacking gels. Molecular weight of proteins in the unknown samples was determined as recommended by the manufacturer of the standards. Protein sequencing: amino acid sequencing, using automated Edman degradation of HPLC purified protein, was performed. The sequence obtained was compared with the known protein sequence of bovine lactoferrin. RESULTS: HPLC analysis of whole bovine tears resulted in a consistent chromatogram. Peak collection was performed to recover a protein from the bovine tear film with chromatogram characteristics nearly identical to purified bovine lactoferrin. Silver-stained SDS-PAGE of this peak revealed a band with molecular mass consistent with bovine lactoferrin (estimated mass of 78 kd). The first 13 amino acid residues of this protein were identical to the amino acid sequence of bovine lactoferrin. CONCLUSION: Analysis of whole bovine tears, using size exclusion HPLC, SDS-PAGE, and amino acid sequencing, provided evidence that bovine tears contain lactoferrin. CLINICAL RELEVANCE: Lactoferrin probably exerts a bacteriostatic effect in bovine tear film. Locally produced lactoferrin may bathe the ocular surface and sequester iron from potential pathogens.

Glomerular ultrastructural lesions of idiopathic cutaneous and renal glomerular vasculopathy of greyhounds.

Idiopathic cutaneous and renal glomerular vasculopathy (CRGV) (Alabama rot) is a potentially fatal disease of unknown etiology that affects the skin and kidneys of racing- and training-age Greyhounds. Ultrastructural examinations were performed on two healthy control Greyhounds and 12 Greyhounds diagnosed with CRGV based on the presence of characteristic, well-demarcated cutaneous ulcers of the extremities (12/12), thrombocytopenia (< 200,000 platelets/dl) (12/12), and acute renal insufficiency (BUN > 40 mg/dl, serum creatinine > 2.0 mg/dl) (7/12). Early glomerular ultrastructural changes included endothelial swelling, detachment, and necrosis; membranous whorl formation; and platelet adhesion and aggregation. Some capillaries were occluded with aggregated platelets, cellular fragments, and fibrin. Later changes included narrowing of capillary lumina and thickening of glomerular capillary walls by subendothelial accumulation of flocculent, amorphous, variable electron-dense material and occasionally erythrocytes, cellular processes, and fibrin. Glomerular endothelial cells were increased in number and plump, with villouslike cytoplasmic projections. Mesangial cell cytoplasmic processes occasionally were interposed between the endothelium and the basement membrane. No etiologic agents or electron-dense deposits typical of immune complexes were observed. Although the specific etiology was not determined, the ultrastructural changes suggest that glomerular endothelial damage is an important early event in the pathogenesis of CRGV.

Growth-condition dependent expression of Pasteurella haemolytica A1 outer membrane proteins, capsule, and leukotoxin.

Pasteurella haemolytica, strain P1148 (biotype A, serotype 1) was grown under iron-rich and iron-restricted conditions both with and without serum, and the outer membrane protein (OMP), capsule, and leukotoxin production studied. OMPs were evaluated by SDS-PAGE and examined by immunoblot to identify antigens recognized by sera from P. haemolytica A1 convalescent and vaccinated cattle. Capsule production was evaluated using fluorescent antibody staining and rapid plate agglutination reaction. Leukotoxin production was measured by neutrophil 51Cr-release assay. Expression of specific OMPs, amount and antigenic character of capsule, and quantity of leukotoxin produced by P. haemolytica A1 varied in response to alterations in the growth media. Immunoblots indicated the immune response of convalescent calves differs from vaccinated calves, and convalescent calves produce antibodies to novel OMPs induced by growth in iron-restricted conditions.

Inhibition of lipopolysaccharide-induced TNF-alpha production by semisynthetic polymyxin-B conjugated dextran.

During episode of severe endotoxemia, concentrations of both lipopolysaccharide and its lipid A-core subfraction are liberated from gram-negative bacteria and become elevated within the systemic circulation. Lipid-A core is the most homogeneous and physiologically toxic segment of the lipopolysaccharide molecule. Polymyxin-B has profound binding avidity for the lipid A-core subfraction of lipopolysaccharide. The mechanism of this binding avidity involves the development of attractive forces between the cationic groups of polymyxin-B and the anionic groups of the lipid A-core moiety of lipopolysaccharide. Complementary attractive forces include hydrophobic interactions which additionally become established between the octylheptanoyl group of polymyxin-B and the saturated carbon chains of the lipid A-core moiety. This paper describes a method for the semisynthetic production of polymyxin-B conjugated dextran in the form of polymyxin-B.ABH.dextran applying the photoreactive crosslinking reagent azidobenzoyl hydrazide (ABH). Molecular design and development of a semisynthetic technique for the conjugation of polymyxin-B to purified dextran fractions was motivated by the pronounced nephrotoxicity associated with this cationic polypeptide antibiotic. Conjugation of polymyxin-B to a relatively large molecular weight carrier compound would increase the overall size of the complex to a degree sufficient to theoretically reduce clearance through glomerular filtration mechanisms. Attributes of such a large molecular weight polymyxin-B conjugated biopharmaceutical would include diminished levels of nephrotoxicity due to a reduction of renal tubular concentrations and a simultaneous prolongation of its intravascular half-life (t (1/2)) and pharmacokinetic profile. Lipopolysaccharide (LPS) binding avidity of polymyxin-B.ABH.dextran was verified by Dot-Blot analysis in conjunction with the application of fluorescein isothiocyanate conjugated E. coli (0.55:B5) LPS (FITC-LPS). Capacity of polymyxin-B.ABH.dextran conjugates to inhibit in vitro LIP-induced synthesis of tumor necrosis factor-alpha (TNF-alpha) was assessed by the application of a tissue culture based biological assay system capable of detecting cytotoxicity mediated by this potent monokine. Semisynthetic conjugates of polymyxin-B.ABH.dextran conjugates (0.6 microns/mL), thereby providing cytoprotectivity (95%; p < or - 0.001. to WEHI 164 clone 13 cell populations relative to untreated reference controls. Since TNF-alpha is currently believed to be the principal endogenous mediator involved in the host's inflammatory response during episodes of endotoxemia, results from these investigations provide a scientific foundation for warranting the elevation of the in vivo efficacy of large molecular weight semisynthetic polymyxin-B conjugates. Results from these investigations may ultimately lead to the application of semisynthetic polymyxin-B.ABH.dextran as a model for the molecular design of semisynthetic production of alternative biopharmaceutical or pharmaceutical agents possessing prophylactic and/or therapeutic efficacy for the management of severe endotoxemia conditions.

Development and evaluation of a mixed-antigen ELISA for serodiagnosis of Actinobacillus pleuropneumoniae serotypes 1, 5, and 7 infections in commercial swine herds.

A mixed-antigen enzyme-linked immunosorbent assay (ELISA) containing serotype-specific polysaccharide antigens from serotypes 1, 5, and 7 of Actinobacillus pleuropneumoniae was developed. This ELISA was evaluated using sera from experimentally infected pigs. With a negative cutoff value of 0.250 (optical density at 405 nm), sensitivity and specificity were determined to be 96% and 99.5%, respectively. The ELISA was further evaluated using sera from commercial swine. Sows and suckling piglets were found to be the best age groups for detection of positive reactors to A. pleuropneumoniae. The mixed-antigen ELISA could be used in a herd health monitoring program for detection of A. pleuropneumoniae infections.

Isolation of an inhibitor of tumor necrosis factor-alpha-mediated cytotoxicity liberated from chemotaxin-stimulated equine white blood cell populations.

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.

Serotype identification of Actinobacillus pleuropneumoniae by arbitrarily primed polymerase chain reaction.

Rapid and accurate determination of the Actinobacillus pleuropneumoniae serotype involved in a disease outbreak is important both in limiting the severity of an outbreak and for tracing the source of the infecting organism. This study describes the use of arbitrarily primed polymerase chain reaction (AP-PCR) as a rapid, precise, and genetically based procedure to identify A. pleuropneumoniae. AP-PCR amplification of bacterial genomic DNA results in specific DNA profiles, which can be used to differentiate currently recognized serotypes. This technique is especially useful for identifying previously nontypeable and serologically cross-reactive A. pleuropneumoniae field isolates. Consecutive passages of isolates on different media, freezing, and subsequent infection of pigs did not alter the AP-PCR genomic profile. We propose the use of M13 and T3-T7 oligodeoxynucleotide primers for diagnostic and epidemiological identification of A. pleuropneumoniae by AP-PCR techniques.