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  • Author or Editor: Kenneth D. Clinkenbeard x
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SUMMARY

Objective

To screen supernatants of Pasteurella haemolytica cultures grown in 4 serum-free culture media for maximal leukotoxin (LKT) production and minimal protein concentration as an optimal source of LKT for purification.

Sample Population

One strain of P haemolytica biotype A serotype 1 originally isolated from the pneumonic lung of a calf.

Procedure

Pasteurella haemolytica was grown in brain-heart infusion (BHI) broth, yeast-tryptone broth, RPMI-1640 medium, and McCoy's modified 5A medium. Culture biomass and protein concentration, LKT activity, and LKT concentration in culture supernatants were measured. Effects of media pH and supplementation with metal cations and glucose on growth rate of P haemolytica and culture supernatant parameters were evaluated.

Results

Pasteurella haemolytica cultivated in BHI broth or RPMI-1640 medium containing 0.1M phosphate (pH 6.8) produced the highest concentrations of LKT. Supplementation of RPMI-1640 medium with 0.36 mM FeCl3 or 1.0 mM MgSO4 further increased specific activity of LKT in culture supernatant, but addition of 1 % glucose did not enhance LKT production. Leukotoxin production in MgSO4-supplemented RPMI-1640 medium was comparable to that in serum protein-supplemented medium.

Conclusions

Although BHI broth was superior to RPMI-1640 medium for P haemolytica growth and LKT production, the higher protein concentration and lower LKT specific activity made BHI broth a less desirable medium, compared with RPMI-1640 medium. Growth rate and LKT production with minimal protein content was optimal in pH 6.8 phosphate-buffered MgSO4-supplemented RPMI-1640 medium. This medium can serve as a source of culture supernatant for purification of LKT. (Am J Vet Res 1998;59:851–855)

Free access
in American Journal of Veterinary Research

SUMMARY

Pasteurella haemolytica Al culture supernatants caused rapid cytolysis (<5 minutes) of isolated bovine platelets as measured by leakage of the cytoplasmic enzyme lactate dehydrogenase (ld). The platelet lytic factor had several features similar to P haemolytica leukotoxin. Like P haemolytica leukotoxin, the platelet lytic factor was produced by P haemolytica during logarithmic growth phase, was heat-labile, and was active against target cells (platelets) from ruminant species (cattle and sheep), but not from non-ruminant species (horses, pigs, and human beings). Additionally, the platelet lytic factor was neutralized with antileukotoxin rabbit serum. The amount of ld leaked by a fixed concentration of bovine platelets was proportional to the amount of toxin added at low toxic doses and became maximal at 88 ± 11% of the total platelet ld activity for high doses of toxin. When a fixed dose of toxin was used and the platelet concentration was varied, ld leakage was initially proportional to the platelet concentration, but plateaued at higher platelet concentrations. The platelet lytic factor required Ca2+ and was inhibited by addition of the Ca2+ chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid. Toxin-mediated platelet damage may be important in thrombi formation and fibrin exudation typically associated with P haemolytica pleuropneumonia of cattle.

Free access
in American Journal of Veterinary Research

Abstract

Case Description—A 2-year-old captive-bred sexually intact female African pygmy hedgehog (Atelerix albiventris) was evaluated because of vague signs of illness including inappetence, weakness, lethargy, and weight loss over a 20-day period.

Clinical Findings—Abnormalities detected via initial clinicopathologic analyses included anemia, thrombocytopenia, leukopenia, hypoproteinemia, and hypoglycemia. Results of a fecal flotation test were negative. Three weeks after the initial evaluation, splenomegaly was detected via palpation and ultrasonography.

Treatment and Outcome—The hedgehog was treated with broad-spectrum antibacterial agents, resulting in an initially favorable response. Fenbendazole was also administered against possible occult parasitic infestation. After 3 weeks of illness, the hedgehog's condition had worsened and supportive care and administration of additional antibacterial agents were instituted. The hedgehog died, and pathologic examinations revealed severe splenomegaly; granulomatous infiltrates were evident in multiple organs, and Histoplasma capsulatum yeasts were detected intralesionally.

Clinical Relevance—Histoplasmosis can develop in a wide range of mammalian species. African pygmy hedgehogs are becoming increasingly popular as exotic pets, and vague signs of illness and splenomegaly are often attributed to hemolymphatic malignancies, which are somewhat common in this species. Practitioners should be aware that similar clinical signs may be associated with histoplasmosis in these animals. Although the hedgehog of this report was confined indoors, it originated from an area where histoplasmosis was endemic; this indicates that the disease should be included as a differential diagnosis for hedgehogs that develop vague signs of illness and are known to originate from such geographic regions.

Full access
in Journal of the American Veterinary Medical Association

SUMMARY

Bovine pulmonary artery endothelial cells (bpaec) were labeled with 3H-arachidonic acid. Exposure of the labeled bpaec to Pasteurella haemolytica lipopolysaccharide (lps) resulted in a time- and dose-dependent release of radioactivity. The release was inhibited by 5 mM indomethacin, but inhibition was not caused by ≤ 500 μM indomethacin or hydrocortisone, which suggests that the release was caused primarily by a mechanism other than cyclooxygenase or phospholipase A2 metabolism of arachidonic acid.

Pasteurella haemolytica lps also caused increased adherence of bovine neutrophils to bpaec through independent effects on both cell types. The increased adherence was inhibited by treatment of either cell type with cycloheximide or actinomycin D prior to lps exposure, indicating that de novo protein synthesis was required in both cell types to promote the lps-induced adherence. Lipopolysaccharide may be an important factor in neutrophil-mediated effects in pneumonic pasteurellosis by causing increased neutrophil adherence and, thus, the vascular sequestration of neutrophils.

Together, these experiments provide additional evidence for the involvement of lps in pneumonic pasteurellosis. Moreover, they provide evidence of lps-induced endothelial activation, which could have broad ramifications in the inflammatory and immune responses of pneumonic pasteurellosis.

Free access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To determine the efficacy of Bdellovibrio bacteriovorus 109J for the treatment of calves with experimentally induced infectious bovine keratoconjunctivitis (IBK).

ANIMALS 12 healthy dairy calves.

PROCEDURES For each calf, a grid keratotomy was performed on both eyes immediately before inoculation with Moraxella bovis hemolytic strain Epp63–300 (n = 11 calves) or nonhemolytic strain 12040577 (1 calf). For each calf inoculated with M bovis Epp63–300, the eyes were randomly assigned to receive an artificial tear solution with (treatment group) or without (control group) lyophilized B bacteriovorus 109J. Six doses of the assigned treatment (0.2 mL/eye, topically, q 48 h) were administered to each eye. On nontreatment days, eyes were assessed and corneal swab specimens and tear samples were collected for bacterial culture. Calves were euthanized 12 days after M bovis inoculation. The eyes were harvested for gross and histologic evaluation and bacterial culture.

RESULTS The calf inoculated with M bovis 12040577 did not develop corneal ulcers. Of the 22 eyes inoculated with M bovis Epp63–300, 18 developed corneal ulcers consistent with IBK within 48 hours after inoculation; 4 of those eyes developed secondary corneal ulcers that were not consistent with IBK. Corneal ulcer size and severity and the time required for ulcer healing did not differ between the treatment and control groups.

CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that B bacteriovorus 109J was not effective for the treatment of IBK; however, the experimental model used produced lesions that did not completely mimic naturally occurring IBK.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To investigate the concentration-dependent effects of Mannheimia haemolytica (formerly Pasteurella haemolytica) leukotoxin (LKT) on apoptosis and oncosis in bovine neutrophils and to examine the role of calcium ions (Ca2+) in LKT-induced apoptosis.

Sample Population—Neutrophils isolated from blood samples obtained from healthy calves.

Procedure—Neutrophil suspensions were exposed to lytic or sublytic dilutions of LKT and then examined by use of transmission electron microscopy (TEM) or gel electrophoresis. Contribution of extracellular Ca2+ to LKT-induced apoptosis was investigated by incubating neutrophils with LKT or control solutions in buffer containing 1 mM CaCl2 or in Ca2+-free buffer containing 1 mM ethylene glycol-bis (b-aminoethyl ether)- N,N-tetraacetic acid (EGTA) prior to diphenyl amine analysis.

Results—Examination by TEM revealed that bovine neutrophils exposed to lytic dilutions of LKT had changes consistent with oncosis, whereas neutrophils exposed to sublytic dilutions of LKT and staurosporin, an inducer of apoptosis, had changes consistent with apoptosis. Effects of sublytic dilutions of LKT on apoptosis were confirmed by gel electrophoresis. Replacement of extracellular Ca2+ with EGTA, a Ca2+ chelator, reduced apoptosis attributable to the calcium ionophore A23187, but it did not have significant effects on apoptosis induced by LKT or staurosporin.

Conclusions and Clinical Relevance—The ability of LKT to cause apoptosis instead of oncosis is concentration- dependent, suggesting that both processes of cell death contribute to an ineffective host-defense response, depending on the LKT concentration in pneumonic lesions. Furthermore, although Ca2+ promotes A23187-induced apoptosis, it is apparently not an essential second messenger for LKT-induced apoptosis. ( Am J Vet Res 2001;62:136–141)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To characterize ultrastructural changes of bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin (LKT).

Sample Population—Partially purified LKT from a wild type P haemolytica A1 strain and inactive pro-LKT from an isogeneic mutant P haemolytica strain. Isolated bovine lymphocytes were obtained from 2 healthy calves.

Procedure—Isolated bovine lymphocytes were incubated with various concentrations of LKT and pro-LKT for 3 hours at 37 C and examined by use of transmission electron microscopy. A cytochemical Klenow DNA fragmentation assay was used to examine lymphocytes for DNA fragmentation.

Results—Lymphocytes incubated with LKT at a high concentration (1.0 toxic U/ml) had ultrastructural evidence of cytoplasmic and nuclear membrane rupture and swelling or lysis of mitochondria. Low concentrations of leukotoxin (0.1 toxic U/ml) induced DNA fragmentation in 80% of lymphocytes. Ultrastructurally, these cells had nuclear membrane blebbing, cytoplasmic vaculation, chromatin condensation, nuclear fragmentation, and membrane-bound apoptotic bodies. Incubation of lymphocytes with LKT at extremely low concentrations (0.001 toxic U/ml) or with pro-LKT did not alter their ultrastructure. Inclusion of 0.5 mM ZnCl2 in the medium blocked leukotoxin-induced ultrastructural changes in bovine lymphocytes.

Conclusions and Clinical Relevance—Low concentrations of LKT induce apoptosis and high concentrations induce oncotic cell lysis in bovine lymphocytes. The ability of low LKT concentrations to induce apoptosis in host leukocytes may allow bacteria to escape host immune surveillance and colonize the host. (Am J Vet Res 2000;61:51–56)

Full access
in American Journal of Veterinary Research

Summary

Growth of Pasteurella haemolytica A1 in RPMI 1640 medium containing 0.5% bovine serum albumin (bsa) for 2.5 hours enhanced culture supernatant leukotoxic activity (30,700 ± 12,900 toxic units/ml, compared with leukotoxic activity of culture supernatants produced in RPMI 1640 medium alone (120 ± 40 toxic units/ml). Gel filtration chromatography of the leukotoxic activity from RPMI 1640 medium supernatants in buffer containing 50 mM NaCl indicated a single leukotoxic activity peak (peak I) eluting near the gel resin molecular mass exclusion limit (estimated molecular mass of approx 8,000 kd). In contrast, culture supernatants produced in RPMI 1640 plus bovine serum albumin medium (RPMI + bsa) had peak I and 2 additional leukotoxic activity peaks (peaks II and III) with estimated molecular mass of approximately 80 and < 30 kd, respectively. All leukotoxic activity peaks were composed of approximately 100-kd molecular mass leukotoxin protomer, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody against leukotoxin. Subjecting culture supernatant leukotoxic activity produced in RPMI + bsa to gel filtration chromatography in buffer containing 500 mM NaCl or 6M urea resulted in detection of only a single leukotoxic activity peak with estimated approximate molecular mass of 250 and 800 kd, respectively. These findings suggest that Phaemolytica exists as a high molecular mass aggregate with low leukotoxic activity which, in the presence of bsa, partially disaggregates to multiple toxin forms with enhanced leukotoxic activity. Some of these leukotoxin forms interact with dextran-based gel resins at low ionic strength.

Free access
in American Journal of Veterinary Research

Abstract

Objective—To develop a repeatable model for studying colonization with streptomycin-resistant Escherichia coli O157:H7 in adult cattle.

Animals—5 adult mixed-breed beef cattle.

Procedures—Cattle were surgically cannulated in the duodenum, treated daily with streptomycin (33 mg/kg) via the duodenal cannula prior to and during experimental colonizations, and colonized with 1010 CFUs of streptomycin-resistant E coli O157:H7 via the duodenal cannula. Colonization of rectal mucus and shedding in feces were monitored. Antimicrobials were administered to eliminate the colonizing strain so that 5 repeated colonization experiments could be performed. A comprehensive analysis of colonization was performed at necropsy.

Results—Streptomycin treatment resulted in improved experimental colonization variables, compared with untreated controls, during initiation (days 2 to 6) and early maintenance (days 7 to 12) of colonization. Elimination of the colonizing strain followed by 5 repeated colonizations in the same animals indicated the repeatability of the protocol. Positive results of bacteriologic culture of feces 7 and 12 days after colonization were obtained in 100% and 84% of samples, respectively, across all animals and trials. At necropsy, highest magnitude recovery was in terminal rectal mucus.

Conclusions and Clinical Relevance—The model was highly repeatable and novel with respect to streptomycin treatment, use of duodenal cannulas, and repeated colonizations of the same animals. Its use in adult cattle, from which most bovine-derived food originates, is critical to the study of preharvest food safety. The findings have implications for understanding intermittency of shedding in the field and for proposed vaccine-based interventions.

Full access
in American Journal of Veterinary Research