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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.

Background: Yersinia pestis initiates infection by parasitism of host macrophages. In response to macrophage infections, intracellular Y. pestis can assume a filamentous cellular morphology which may mediate resistance to host cell innate immune responses. We previously observed the expression of Y. pestis tellurite resistance proteins TerD and TerE from the terZABCDE operon during macrophage infections. Others have observed a filamentous response associated with expression of tellurite resistance operon in Escherichia coli exposed to tellurite. Therefore, in this study we examine the potential role of Y. pestis tellurite resistance operon in filamentous cellular morphology during macrophage infections., Principal Findings: In vitro treatment of Y. pestis culture with sodium tellurite (Na2TeO3) caused the bacterial cells to assume a filamentous phenotype similar to the filamentous phenotype observed during macrophage infections. A deletion mutant for genes terZAB abolished the filamentous morphologic response to tellurite exposure or intracellular parasitism, but without affecting tellurite resistance. However, a terZABCDE deletion mutant abolished both filamentous morphologic response and tellurite resistance. Complementation of the terZABCDE deletion mutant with terCDE, but not terZAB, partially restored tellurite resistance. When the terZABCDE deletion mutant was complemented with terZAB or terCDE, Y. pestis exhibited filamentous morphology during macrophage infections as well as while these complemented genes were being expressed under an in vitro condition. Further in E. coli, expression of Y. pestis terZAB, but not terCDE, conferred a filamentous phenotype., Conclusions: These findings support the role of Y. pestis terZAB mediation of the filamentous response phenotype; whereas, terCDE confers tellurite resistance. Although the beneficial role of filamentous morphological responses by Y. pestis during macrophage infections is yet to be fully defined, it may be a bacterial adaptive strategy to macrophage associated stresses.

The γ-proteobacterium Francisella tularensis causes seasonal tick-transmitted tularemia outbreaks in natural rabbit hosts and incidental infections in humans in the south-central United States. Although Dermacentor variabilis is considered a primary vector for F. tularensis, Amblyomma americanum is the most abundant tick species in this endemic region. A systematic study of F. tularensis colonization of A. americanum was undertaken to better understand its potential to serve as an overwintering reservoir for F. tularensis and as a bridging vector for human infections. Colony-reared A. americanum were artificially fed F. tularensis subspecies holarctica strain LVS via glass capillaries and colonization levels determined. Capillary-fed larva and nymph were initially infected with 10(4) CFU/tick which declined prior to molting for both stages, but rebounded post-molting in nymphs and persisted in 53% at 10(3) to 10(8) CFU/nymph at 168 days post-capillary feeding (longest sampling time in the study). In contrast, only 18% of adults molted from colonized nymphs maintained LVS colonization at 10(1) to 10(5) CFU/adult at 168 days post-capillary feeding (longest sampling time). For adults, LVS initially colonized the gut and disseminated to salivary glands by 24 h and had an ID50 of <5CFU in mice. Francisella tularensis infected the ovaries of gravid females, but transmission to eggs was infrequent and transovarial transmission to hatched larvae was not observed. The prolonged persistence of F. tularensis in A. americanum nymphs supports A. americanum as an overwintering reservoir for F. tularensis from which seasonal epizootics may originate; however, although the rapid dissemination of F. tularensis from gut to salivary glands in adults A. americanum is compatible with intermittent feeding adult males acting as bridging vectors for incidental F. tularensis infections of humans, acquisition of F. tularensis by adults may be unlikely based on adult feeding preference for larger mammals which are not involved in maintenance of sylvatic tularemia.

One of the objectives of the National Institutes of Allergy and Infectious Diseases (NIAID) Biodefense Program is to identify or develop broad-spectrum antimicrobials for use against bioterrorism pathogens and emerging infectious agents. As a part of that program, our institution has screened the 10 000-compound MyriaScreen Diversity Collection of high-purity druglike compounds against three NIAID category A and one category B priority pathogens in an effort to identify potential compound classes for further drug development. The effective use of a Clinical and Laboratory Standards Institute-based high-throughput screening (HTS) 96-well-based format allowed for the identification of 49 compounds that had in vitro activity against all four pathogens with minimum inhibitory concentration values of ≤16 µg/mL. Adaptation of the HTS process was necessary to conduct the work in higher-level containment, in this case, biosafety level 3. Examination of chemical scaffolds shared by some of the 49 compounds and assessment of available chemical databases indicates that several may represent broad-spectrum antimicrobials whose activity is based on novel mechanisms of action.

Background: Yersinia pestis causes severe disease in natural rodent hosts, but mild to inapparent disease in certain rodent predators such as dogs. Y. pestis initiates infection in susceptible hosts by parasitizing and multiplying intracellularly in local macrophages prior to systemic dissemination. Thus, we hypothesize that Y. pestis disease severity may depend on the degree to which intracellular Y. pestis overcomes the initial host macrophage imposed stress., Methodology/principal Findings: To test this hypothesis, the progression of in vitro infection by Y. pestis KIM62053.1+ of mouse splenic and RAW264.7 tissue culture macrophages and dog peripheral blood-derived and DH82 tissue culture macrophages was studied using microscopy and various parameters of infection. The study showed that during the early stage of infection, intracellular Y. pestis assumed filamentous cellular morphology with multiple copies of the genome per bacterium in both mouse and dog macrophages. Later, in mouse macrophages, the infection elicited spacious vacuolar extension of Yersinia containing vacuoles (YCV), and the filamentous Y. pestis reverted to coccobacillary morphology with genomic equivalents approximately equaling colony forming units. In contrast, Y. pestis infected dog macrophages did not show noticeable extension of YCV, and intracellular Y. pestis retained the filamentous cellular morphology for the entire experiment in DH82 cells or were killed by blood-derived macrophages. In addition, during the later stage of infection, Y. pestis infected mouse macrophages exhibited cell lysis whereas dog macrophages did not., Conclusion/significance: Overall, these results support our hypothesis that Y. pestis in mouse macrophages can overcome the initial intracellular stress necessary for subsequent systemic infection. However, in dogs, failure of Y. pestis to overcome macrophage imposed stress may result in mild or in apparent disease in dogs.

Background: The γ-proteobacterium Francisella tularensis is the etiologic agent of seasonal tick-transmitted tularemia epizootics in rodents and rabbits and of incidental infections in humans. The biology of F. tularensis in its tick vectors has not been fully described, particularly with respect to its quanta and duration of colonization, tissue dissemination, and transovarial transmission. A systematic study of the colonization of Dermacentor variabilis by the F. tularensis subsp. holarctica live vaccine strain (LVS) was undertaken to better understand whether D. variabilis may serve as an inter-epizootic reservoir for F. tularensis., Methodology/principal Findings: Colony-reared larva, nymph, and adult D. variabilis were artificially fed LVS via glass capillary tubes fitted over the tick mouthparts, and the level of colonization determined by microbial culture. Larvae and nymphs were initially colonized with 8.8 ± 0.8 × 10(1) and 1.1 ± 0.03 × 10(3) CFU/tick, respectively. Post-molting, a significant increase in colonization of both molted nymphs and adults occurred, and LVS persisted in 42% of molted adult ticks at 126 days post-capillary tube feeding. In adult ticks, LVS initially colonized the gut, disseminated to hemolymph and salivary glands by 21 days, and persisted up to 165 days. LVS was detected in the salivary secretions of adult ticks after four days post intra-hemocoelic inoculation, and LVS recovered from salivary gland was infectious to mice with an infectious dose 50% of 3 CFU. LVS in gravid female ticks colonized via the intra-hemocoelic route disseminated to the ovaries and then to the oocytes, but the pathogen was not recovered from the subsequently-hatched larvae., Conclusions/significance: This study demonstrates that D. variabilis can be efficiently colonized with F. tularensis using artificial methods. The persistence of F. tularensis in D. variabilis suggests that this tick species may be involved in the maintenance of enzootic foci of tularemia in the central United States.

The objective of this study was to determine the potential of Bdellovibrio bacteriovorus 109J as an alternative non-chemotherapeutic treatment of infectious bovine keratoconjunctivitis (IBK). To accomplish this, various parameters of B. bacteriovorus predation of Moraxella bovis were determined in vitro. Initial passage of B. bacteriovorus using M. bovis as prey required 10 d for active cultures to develop compared with 2 d for culture on normal Escherichia coli prey; however by the 5th passage, time to active predatory morphology was reduced to 2 d. This high passage B. bacteriovorus culture [1 × 10(10) plaque forming units (PFU)/mL] killed 76% of M. bovis [1 × 10(7) colony forming units (CFU)/mL] present in suspension broth in a 4 h assay. The minimal level of M. bovis supporting B. bacteriovorus predation was 1 × 10(4) CFU/mL. To assess the ability of B. bacteriovorus to kill M. bovis on an epithelial surface mimicking IBK, an in vitro assay with Madin-Darby bovine kidney (MDBK) cells inoculated with 4 × 10(7) CFU/mL M. bovis was used. Treatment with a B. bacteriovorus suspension (1.6 × 10(11) PFU/mL) decreased adherence of M. bovis to MDBK cells by 6-fold at 12 h of treatment, as well as decreased the number of unattached M. bovis cells by 1.4-fold. This study demonstrates that B. bacteriovorus has potential as an effective biological control of M. bovis at levels likely present in IBK-infected corneal epithelia and ocular secretions.

The serine protease coagulation factor thrombin functions primarily in hemostasis, but is also involved in atherosclerosis, thromboembolic disease, cancer and inflammatory disease. Direct measurement of coagulation proteins including thrombin in plasma samples poses a significant challenge because of lack of specific probes and low thrombin concentrations. In addition, high plasma protein concentrations in samples can result in high backgrounds. These challenges were overcome using a bi-cell surface plasmon resonance (SPR) spectrometer with an immobilized thrombin aptamer to measure thrombin in samples passed through a low volume flow cell. For thrombin in Tris-EDTA buffer, the limit of detection (LOD) was 25 nM. Coefficient of variation (CV) for detection of 50 nM was 12.2% and 12.4% for intra and inter-day measurements respectively. This detection was specific for both thrombin aptamer and for thrombin. Using serum samples spiked with thrombin, the LOD was 50 nM with a linear range of detection from 50 nM to 200 nM. However use of serum samples was associated with consistent, low-level background drift. The contributions of nonspecific protein absorption onto the sensor surface and sample flow speed were assessed, and strategies to reduce this background drift were explored. We conclude that the bi-cell SPR platform with an aptamer capture probe can be employed as a highly sensitive real-time, label-free biosensor for the detection of coagulation factors in plasma samples., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Yersinia pestis initiates infection as a facultative intracellular parasite in host macrophages; however, little is known about the efficacy of antibiotics commonly used to treat human plague against intracellular Y. pestis. Intracellular minimal bactericidal concentrations (MBCs) were determined using a high-throughput broth microdilution assay in which human THP-1 macrophage-like cells were infected with Y. pestis strain KIM6-2053.1+ and exposed to 2-fold serial dilutions of antibiotics for 24 h in 96-well plates. The numbers of CFU, upon which minimal bactericidal concentrations were based, were determined by counting "microcolonies" in wells of 96-well plates following lysis of tissue culture cells to release surviving Y. pestis, replica dilution, and plating in soft tryptic soy broth agar. For THP-1 cells, streptomycin and ciprofloxacin had comparable efficacies for intra- and extracellular Y. pestis, but the MBCs for chloramphenicol, gentamicin, doxycycline, and amoxicillin were two-, three-, four-, and five 2-fold serial dilutions greater, respectively, for intracellular than for extracellular Y. pestis. During the initial stage of plague, intracellular Y. pestis may be less susceptible to antibiotic killing by particular antibiotics recommended for treatment of plague, such as gentamicin or doxycycline, whereas others, such as streptomycin and ciprofloxacin, may have similar efficacies against extracellular or intracellular Y. pestis. This may be of particular importance in the selection of antibiotics for prophylactic treatment in the case of a bioterrorism event.

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.

Background: Bacillus spores are notoriously resistant to unfavorable conditions such as UV radiation, gamma-radiation, H2O2, desiccation, chemical disinfection, or starvation. Bacillus pumilus SAFR-032 survives standard decontamination procedures of the Jet Propulsion Lab spacecraft assembly facility, and both spores and vegetative cells of this strain exhibit elevated resistance to UV radiation and H2O2 compared to other Bacillus species., Principal Findings: The genome of B. pumilus SAFR-032 was sequenced and annotated. Lists of genes relevant to DNA repair and the oxidative stress response were generated and compared to B. subtilis and B. licheniformis. Differences in conservation of genes, gene order, and protein sequences are highlighted because they potentially explain the extreme resistance phenotype of B. pumilus. The B. pumilus genome includes genes not found in B. subtilis or B. licheniformis and conserved genes with sequence divergence, but paradoxically lacks several genes that function in UV or H2O2 resistance in other Bacillus species., Significance: This study identifies several candidate genes for further research into UV and H2O2 resistance. These findings will help explain the resistance of B. pumilus and are applicable to understanding sterilization survival strategies of microbes.

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 10(10) 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.

The gamma-proteobacterium Francisella tularensis is one of the most infectious human pathogens, and the highly virulent organism F. tularensis subsp. tularensis (type A) and less virulent organism F. tularensis subsp. holarctica (type B) are most commonly associated with significant disease in humans and animals. Here we report the complete genome sequence and annotation for a low-passage type B strain (OSU18) isolated from a dead beaver found near Red Rock, Okla., in 1978. A comparison of the F. tularensis subsp. holarctica sequence with that of F. tularensis subsp. tularensis strain Schu4 (P. Larsson et al., Nat. Genet. 37:153-159, 2005) highlighted genetic differences that may underlie different pathogenicity phenotypes and the evolutionary relationship between type A and type B strains. Despite extensive DNA sequence identity, the most significant difference between type A and type B isolates is the striking amount of genomic rearrangement that exists between the strains. All but two rearrangements can be attributed to homologous recombination occurring between two prominent insertion elements, ISFtu1 and ISFtu2. Numerous pseudogenes have been found in the genomes and are likely contributors to the difference in virulence between the strains. In contrast, no rearrangements have been observed between the OSU18 genome and the genome of the type B live vaccine strain (LVS), and only 448 polymorphisms have been found within non-transposase-coding sequences whose homologs are intact in OSU18. Nonconservative differences between the two strains likely include the LVS attenuating mutation(s).

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.

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 Phaemolytica 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.

Pasteurella haemolytica, the causative agent of shipping fever pneumonia in cattle, produces a leukotoxin (LKT) which lyses ruminant leukocytes with high efficiency but is reputed to not affect leukocytes from nonruminant species. In this study, we tested the supposition that LKT binding correlates positively with susceptibility to intoxication of susceptible isolated bovine lymphocytes and lymphoma tissue culture cells (BL3 cells) and negatively with reputed nonsusceptible equine, porcine, and canine lymphocytes and human lymphoid tissue culture cells (Raji cells). Bovine lymphocytes and BL3 cells were highly susceptible to LKT intoxication, exhibiting both substantial increase in intracellular Ca(2+) concentration and marked leukolysis. Exposure of reputed LKT-nonsusceptible porcine lymphocytes and Raji cells to LKT caused a slightly increased intracellular Ca(2+) concentration but no leukolysis. No LKT effect was detected for equine and canine lymphocytes. LKT bound to lymphoid cells from all species tested. Intact 102-kDa LKT was recovered from exposed isolated lymphoid cell membranes. Pro-LKT acylation was not required for LKT binding to BL3 cells. LKT binding was rapid, with maximal binding occurring by 3 min, and was proportional to the LKT concentration in the range 0.04 to 4.0 microg/ml. For this LKT concentration range, BL3 cells bound more LKT than did porcine lymphocytes or Raji cells, suggesting that LKT binds to BL3 cells with higher affinity than to porcine lymphocytes or Raji cells. Above 4.0 microg/ml, LKT demonstrated saturable binding to BL3 cells. Neutralizing anti-LKT monoclonal antibody (MAb) MM601 diminished LKT binding to BL3 by 36% while decreasing leukolysis by 81%. In contrast, MM601 did not diminish LKT binding to Raji cells. Pretreatment of target cells with 120 microg of protease K per ml diminished LKT binding to BL3 cells by 75%, with only a 25% decrease in leukolysis. However, pretreatment with 150 microg of protease K per ml abolished the remaining 25% of LKT binding and 75% leukolysis. Therefore, P. haemolytica LKT binds rapidly to susceptible and to reputed nonsusceptible lymphoid cells. LKT binding resulting in species-specific leukolysis was characterized by high affinity, inhibition by MAb MM601, and relative resistance to protease K pretreatment of lymphoid cells. Two types of LKT binding to lymphoid cells are proposed. High-affinity binding leads to efficient leukolysis. In some lymphoid cells from reputed LKT-nonsusceptible species, low-affinity LKT binding may cause a low-efficiency increase in the intracellular Ca(2+) concentration without leading to leukolysis.

The effects of Pasteurella haemolytica leukotoxin (LKT) on the activity of phospholipase D (PLD) and the regulatory interaction between PLD and phospholipase A(2) (PLA(2)) were investigated in assays using isolated bovine neutrophils labeled with tritiated phospholipid substrates of the two enzymes. Exposure of [(3)H]lysophosphatidylcholine-labeled neutrophils to LKT caused concentration- and time-dependent production of phosphatidic acid (PA), the product of PLD. LKT-induced generation of PA was dependent on extracellular calcium. Both production of PA and metabolism of [(3)H]-arachidonate ([(3)H]AA)-labeled phospholipids by PLA(2) were inhibited when ethanol was used to promote the alternative PLD-mediated transphosphatidylation reaction, resulting in the production of phosphatidylethanol rather than PA. The role of PA in regulation of PLA(2) activity was then confirmed by means of an add-back experiment, whereby addition of PA in the presence of ethanol restored PLA(2)-mediated release of radioactivity from neutrophil membranes. Considering the involvement of chemotactic phospholipase products in the pathogenesis of pneumonic pasteurellosis, development and use of anti-inflammatory agents that inhibit LKT-induced activation of PLD and PLA(2) may improve therapeutic management of the disease.

The presence of lipopolysaccharide (LPS) in gram-negative bacterial repeats-in-toxin (RTX) toxin preparations, as well as the harsh conditions required to remove it, suggests that LPS may complex with RTX toxins. Concentrated culture supernatant (CCS) preparations of the RTX toxin Pasteurella haemolytica leukotoxin (LKT) contained LKT and LPS as the most prominent components, with LKT and LPS constituting approximately 30 and 50% of the density of the silver-stained fraction on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. CCS LKT contained 3.69 +/- 0.46 mg of LPS per mg of protein, which was estimated to indicate an LPS/LKT molar ratio of approximately 60:1. Subjection of the CCS LKT to preparative SDS-PAGE resulted in separation of LPS from LKT as detected by silver-stained analytical SDS-PAGE; however, the LKT fraction (SDS-PAGE LKT) contained significant endotoxin activity as detected by the Limulus amebocyte lysate assay. Subjection of the SDS-PAGE LKT to a second preparative SDS-PAGE run resulted in a reduction of the LPS/LKT molar ratio to 1:20. The target cell specificity of LKT for bovine leukocytic cells was retained by the SDS-PAGE LKT, and isolated LPS at comparable concentrations to that in CCS LKT exhibited no leukolytic activity. Addition of isolated LPS back to SDS-PAGE LKT resulted in reconstitution of an LPS-LKT complex. Immediately following reconstitution of the LPS-LKT complex, there was minimal change in leukolytic activity of the complex, but following 9.5 h at temperatures from -135 to 37 degrees C, the LPS-LKT complex exhibited increased leukolytic activity and thermal stability compared to SDS-PAGE LKT. Therefore, it appears that LPS complexes with LKT, resulting in enhanced and stabilized leukolytic activity.

A Pasteurella haemolytica mutant incapable of producing leukotoxin was created by allelic replacement. Concentrated culture supernatants from wild-type P. haemolytica, but not from the mutant, contained the 102-kDa leukotoxin protein and lysed bovine lymphoma cells and sheep erythrocytes. Wild-type P. haemolytica demonstrated the typical beta-hemolytic phenotype on sheep and rabbit blood agar, whereas the mutant did not.

Exposure of bovine pulmonary artery endothelial cells to Pasteurella haemolytica lipopolysaccharide caused severe morphologic changes. Initially, there was dilatation of the rough endoplasmic reticulum and mitochondrial swelling followed by cell retraction, membrane bleb formation, and cell detachment. The affected endothelial cells had severe membrane damage resulting in the leakage of lactate dehydrogenase. Indomethacin in concentrations of 0.5 mM or greater caused marked decreases in the lipopolysaccharide-induced leakage of lactate dehydrogenase. Indomethacin at 5 mM also caused a marked reduction of the lipopolysaccharide-induced morphologic changes resulting in apparent maintenance of the monolayer integrity for 8 hours versus 1 hour in the lipopolysaccharide-treated control. A marked decrease in the cell and nuclear membrane effects resulted, but the rough endoplasmic reticulum dilatation and mitochondrial changes proceeded. These results indicate that indomethacin does not prevent lipopolysaccharide binding but interferes with later events in lipopolysaccharide-induced cytotoxicity in the bovine pulmonary endothelial cell. The concentration of indomethacin required to produce this inhibition suggests that the primary mechanism is not cyclooxygenase inhibition.

Exposure of isolated bovine neutrophils to partially purified Pasteurella haemolytica leukotoxin caused increased synthesis of leukotriene B4 (LTB4) but not thromboxane B2 (TXB2) from endogenous arachidonic acid. Synthesis of LTB4 was closely correlated with leukotoxin-induced neutrophil lysis. At low toxin concentrations, LTB4 production lagged behind leukotoxin-induced neutrophil lysis over a 3-h period. The neutralizing monoclonal antileukotoxin antibody MM601 neutralized both leukotoxin-induced neutrophil lysis and LTB4 synthesis. Both leukotoxin-induced neutrophil lysis and LTB4 synthesis were Ca(2+)-dependent. When leukotoxin-induced LTB4 synthesis from exogenous arachidonic acid was examined, significant LTB4 synthesis occurred at 5 min of leukotoxin exposure, which was before leukotoxin-induced lysis developed. Leukotoxin-induced LTB4 synthesis from endogenous arachidonic acid appears to require leukotoxin-induced plasma membrane damage (occurring during neutrophil lysis), whereas LTB4 synthesis from exogenous arachidonic acid is initiated rapidly and occurs in the absence of plasma membrane damage.

Bovine erythrocytes (RBCs) exposed to Moraxella bovis culture supernatants exhibited rapid leakage of intracellular K+ (95% in 10 min), slower cell swelling (1.20-fold increase in mean corpuscular volume in 20 min), and subsequent lysis (76% leakage of hemoglobin in 25 min). Incubation media made hypertonic by the addition of 75 mM carbohydrates with molecular diameters of 0.72 to 1.32 nm prevented hemolysin-induced RBC swelling, but incubation media made hypertonic by the addition of carbohydrates with molecular diameters of less than 0.72 nm did not protect against hemolysin-induced RBC swelling. Raffinose (75 mM; molecular diameter, 1.14 nm) did not block hemolysin-induced K+ leakage but did block hemolysis. These findings support the hypothesis that hemolysin-induced lysis occurs by colloid-osmotic swelling and are compatible with M. bovis hemolysin acting as a pore-forming cytolysin. Assuming that M. bovis hemolysin acts as a transmembrane molecular sieve, then the functional size of the hemolysin transmembrane pores in bovine RBCs is approximately 0.9 nm, the molecular size of sucrose. Hemolytic activity was inhibited by the Ca2+ chelator ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), but hemolysin-induced K+ leakage was not affected by EGTA.

Pasteurella haemolytica is represented by two biotypes (A and T), 15 serotypes, and numerous untypable strains. Specific biotypes and serotypes are associated with fibrinous pleuropneumonia (pneumonic pasteurellosis) in cattle, sheep, and goats, septicemic pasteurellosis in lambs, and mastitis in ewes. Four virulence factors have been associated with P. haemolytica: fimbriae, a polysaccharide capsule, endotoxin [lipopolysaccharide (LPS)], and leukotoxin (LKT). The interactions of these virulence factors with components of the pulmonary alveolus are discussed as a model for the pathogenesis of pasteurellosis. Fimbriae on P. haemolytica may enhance colonization of the upper respiratory tract. The capsule of P. haemolytica varies in composition among serotypes. It inhibits complement-mediated serum killing as well as phagocytosis and intracellular killing of P. haemolytica. The capsule enhances neutrophil directed migration and adhesion of P. haemolytica to alveolar epithelium. Pasteurella haemolytica LPS can alter bovine leukocyte functions, by dose-dependent inhibition or augmentation; it is directly toxic to bovine endothelium; it modifies cardiopulmonary hemodynamics; and it elevates circulatory prostanoids, serotonin, cAMP, and cGMP. Leukotoxin is produced by all known serotypes and many untypable strains. Leukotoxin is a poreforming cytolysin that affects ruminant leukocytes and platelets by altering function at low levels but causing lysis at high levels.

Antigens associated with whole Pasteurella haemolytica biotype A serotype 1, a capsular carbohydrate-protein extract of the organism, and P. haemolytica leukotoxin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antigens of the electrophoresed preparations were detected by Western blotting (immunoblotting) with sera from cattle which were either nonvaccinated or vaccinated with live or killed P. haemolytica vaccines and had variable degrees of resistance to experimental pneumonic pasteurellosis. Distinct, easily recognizable antigens of these preparations were identified, and the antibody responses to these antigens were quantified by densitometry. To determine their importance to disease resistance, we then compared antibody responses with experimental lesion scores. Antibody reactivity to surface antigens which were significantly correlated with resistance and present in two or more of the preparations were detected at 86, 66, 51, 49, 34, 31, and 16 kilodaltons (kDa). Of these, antibody responses to antigens at 86, 49, and 31 kDa appeared most important based on their concentration and significance levels. Antibody reactivity to leukotoxin antigens which were significantly correlated with resistance and common with important surface antigens were detected at 86, 66, and 49 kDa. Antibody responses to unique leukotoxin antigens which were significantly correlated with resistance were present at 92 and 58 kDa.

Homogeneous liver 3-hydroxy-3-methylglutaryl coenzyme A synthase, which catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA, also carries out: (a) a rapid transacetylation from acetyl-CoA to 31-dephospho-CoA and (b) a slow hydrolysis of acetyl-CoA to acetate and CoA. Transacetylation and hydrolysis occur at 50 and 1 percent, respectively, the rate of the synthasecatalyzed condensation reaction. It appears that an acetyl-enzyme intermediate is involved in the transacetylase and hydrolase reactions of 3-hydroxy-3-methylglutaryl-CoA synthase, as well as in the over-all condensation process. Covalent binding to the enzyme of a [14C]acetyl group contributed by [1(-14)C]acetyl-CoA is indicated by migration of the [14C]acetyl group with the dissociated synthase upon electrophoresis in dodecyl sulfate-urea and by precipitation of [14C]acetyl-enzyme with trichloroacetic acid. At 0 degrees and a saturating level of acetyl-CoA, the synthase is rapidly (less than 20 s) acetylated yielding 0.6 acetyl group/enzyme dimer. Performic acid oxidation completely deacetylates the enzyme, suggesting the site of acetylation to be a cysteinyl sulfhydryl group. Proteolytic digestion of [14C]acetyl-S-enzyme under conditions favorable for intramolecular S to N acetyl group transfer quantitatively liberates a labeled derivative with a [14C]acetyl group stable to performic acid oxidation. The labeled oxidation product is identified as N-[14C]acetylcysteic acid, thus demonstrating a cysteinyl sulfhydryl group as the original site of acetylation. The ability of the acetylated enzyme, upon addition of acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA indicates that the acetylated cysteine residue is at the catalytic site.

Pasteurella haemolytica A1 leukotoxin causes rapid (5 to 15 min) leakage of intracellular K+ and cell swelling and slower (15 to 60 min), Ca2+-dependent formation of large plasma membrane defects (congruent to 100 nm) and leakage of lactate dehydrogenase from bovine lymphoma cells (BL3 cells) (K. D. Clinkenbeard, D. A. Mosier, A. L. Timko, and A. W. Confer, Am. J. Vet. Res., in press). Incubation of BL3 cells in medium made hypertonic by inclusion of 75 mM sucrose blocked leukotoxin-induced cell swelling, formation of large plasma membrane defects, and leakage of lactate dehydrogenase but did not block leukotoxin-induced leakage of intracellular K+. Carbohydrates with molecular weights less than that of sucrose, e.g., mannitol, did not block leukotoxin-induced cell swelling of BL3 cells. Increasing the concentration of mannitol to twice that of sucrose still resulted in no protective effect. Assuming that leukotoxin acts as a transmembrane molecular sieve, then the functional transmembrane pore size formed by leukotoxin in BL3 cells is slightly less than the size of sucrose, i.e., 0.9 nm. Exposure of BL3 cells to leukotoxin for 15 or 45 min followed by the addition of hypertonic sucrose to the incubation medium reversed leukotoxin-induced cell swelling and prevented further leakage of lactate dehydrogenase. Leukotoxin-induced leakage of lactate dehydrogenase required both cell swelling and Ca2+-dependent processes. The Ca2+-dependent steps can occur before or concurrent with cell swelling.