Bighorn sheep beta2-integrin LFA-1 serves as a receptor for Mannheimia haemolytica leukotoxin

Mannheimia haemolytica is an important cause of pneumonia in bighorn sheep (BHS; Ovis canadensis). Leukotoxin (Lkt), the primary virulence determinant of M. haemolytica, induces cytolysis of all subsets of leukocytes. Previously, we have shown that CD18, the beta subunit of beta2-integrins, mediates Lkt-induced cytolysis. However, it is not clear whether CD18 of all three beta2-integrins, LFA-1, Mac-1, and CR4, mediates Lkt-induced cytolysis. The objective of this study was to determine whether BHS LFA-1 (CD11a/CD18) serves as a receptor for Lkt. Plasmids encoding cDNA for BHS CD11a and CD18 were cotransfected into Lkt-resistant HEK-293 cells. Flow cytometric analysis of transfectants confirmed cell surface expression of BHS LFA- 1, Lkt-LFA-1 binding and Lkt-induced intra-cellular calcium elevation. More importantly, the transfectants were efficiently lysed by Lkt in a concentration-dependent manner. Collectively, these results indicate that BHS LFA-1 serves as a functional receptor for M. haemolytica Lkt.     

The leukotoxin of Pasteurella haemolytica binds to β2 integrins on bovine leukocytes

The putative receptor proteins of Pasteurella haemolytica leukotoxin were isolated from bovine polymorphonuclear neutrophil lysate by affinity chromatography on a leukotoxin-specific monoclonal antibody column to which the leukotoxin was pre-bound. SDS-PAGE of the purified proteins showed four bands at 180 kDa, 170 kDa, 150 kDa and 95 kDa, in addition to the expected 102-kDa leukotoxin band and a series of bands with molecular masses lower than 102 kDa representing the disintegrated leukotoxin. N-terminal amino acid sequencing of the 170-kDa band showed homology with human and murine CD11b. The purified proteins reacted specifically with monoclonal antibodies specific for CD11a, CD11b, CD11c (the alpha chains of beta(2) integrins), and CD18 (the beta chain of beta(2) integrins). Pre-incubation of polymorphonuclear neutrophils with a monoclonal antibody specific for CD18 reduced the cytotoxicity of the leukotoxin to the cells. These results indicate that the leukotoxin binds to the beta(2) integrins on bovine leukocytes, very likely via CD18.     

Isolation of Pasteurella haemolytica from tonsillar biopsies of Rocky Mountain bighorn sheep

Isolations of Pasteurella haemolytica were compared from tonsillar biopsies versus nasal passages for 29 free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from central Idaho. Overall, P. haemolytica was isolated from 11 (38%) of 29 sheep. Two (18%) of the 11 positive samples were from only nasal passages compared to eight (73%) from tonsillar biopsies. Pasteurella haemolytica biotype T was isolated from tonsils of nine sheep and from nasal biopsies. Pasteurella haemolytica biotype T was isolated from tonsils of nine sheep and from nasal passages of only one sheep. Two sheep were positive for P. haemolytica biotype A from nasal passages. Culturing tonsillar biopsies as compared to nasal swab samples was a more reliable technique in detecting P. haemolytica, especially biotype T, in bighorn sheep.     

Detecting nonhemolytic Pasteurella haemolytica infections in healthy Rocky Mountain bighorn sheep (Ovis canadensis canadensis): influences of sample site and handling

Effects of sampling procedures on ability to culture Pasteurella spp. from Rocky Mountain bighorn sheep (Ovis canadensis canadensis) were examined experimentally. Sample site influenced (P less than 0.0001) recovery of P. haemolytica in adult bighorn sheep. We isolated nonhemolytic P. haemolytica from 18 of 19 tonsillar swabs and 18 of 19 tonsillar biopsies from adult sheep, yet only four of 19 nasal swabs yielded isolates. Sample handling also affected (P less than 0.0001) recovery of P. haemolytica. Nonhemolytic P. haemolytica was cultured from 14 of 19 tonsillar swabs plated directly onto blood agar, but from only two of 19 swabs stored for 24 hr in modified Stuart's medium. We detected nonhemolytic P. haemolytica at least once in bronchial aspirates from four and in nasal swabs from three of six bighorn lambs. Based on direct cultures of tonsillar swabs and/or biopsies, all 26 bighorn sheep (seven lambs, 19 adults) sampled were infected with nonhemolytic P. haemolytica; only two lambs developed pneumonia during the study period. Thirty-four of 37 nonhemolytic P. haemolytica isolates tested were biotype T; three were biotype A. Serotypes 3; 4; 3, 4 and 3, 4, 10 were identified in a subsample of 17 isolates. Our data suggest tonsillar swabs or biopsies plated directly onto blood agar and incubated immediately offer the greatest probability of recovering nonhemolytic P. haemolytica from health bighorn sheep.     

Sharing of Pasteurella spp. between free-ranging bighorn sheep and feral goats

Pasteurella spp. were isolated from feral goats and free-ranging bighorn sheep (Ovis canadensis canadensis) in the Hells Canyon National Recreation Area bordering Idaho, Oregon, and Washington (USA). Biovariant 1 Pasteurella haemolytica organisms were isolated from one goat and one of two bighorn sheep found in close association. Both isolates produced leukotoxin and had identical electrophoretic patterns of DNA fragments following cutting with restriction endonuclease HaeIII. Similarly Pasteurella multocida multocida a isolates cultured from the goat and one of the bighorn sheep had D type capsules, serotype 4 somatic antigens, produced dermonecrotoxin and had identical HaeIII electrophoretic profiles. A biovariant U(beta) P.haemolytica strain isolated from two other feral goats, not known to have been closely associated with bighorn sheep, did not produce leukotoxin but had biochemical utilization and HaeIII electrophoretic profiles identical to those of isolates from bighorn sheep. It was concluded that identical Pasteurella strains were shared by the goats and bighorn sheep. Although the direction of transmission could not be established, evidence suggests transmission of strains from goats to bighorn sheep. Goats may serve as a reservoir of Pasteurella strains that may be virulent in bighorn sheep; therefore, goats in bighorn sheep habitat should be managed to prevent contact with bighorn sheep. Bighorn sheep which have nose-to-nose contact with goats should be removed from the habitat.     

Immunologic responses of domestic and bighorn sheep to a multivalent Pasteurella haemolytica vaccine

The efficacy of a Pasteurella haemolytica vaccine (serotypes A1, A2, and T10) to induce humoral antibodies and alter colonization of the upper respiratory tract by related P. haemolytica spp. strains was evaluated in 10 bighorn (Ovis canadensis canadensis) and 10 domestic (Ovis aries) sheep. Sheep of each species were divided into five pairs based on age and history of respiratory disease. One sheep in each pair was vaccinated twice 2 wk apart with 2 ml of vaccine (VAC group) and the remaining animals (NV group) were injected with 2 ml of sterile saline. Mild, transient lameness was the only observed adverse effect. Blood sera from the sheep were tested for agglutinating antibodies against whole cells of A1, A2, and T10 and for leukotoxin neutralizing antibodies. Antibody titers were expressed as the reciprocal log2 of the highest reactive dilutions. Domestic sheep > 1-yr-old and two bighorn sheep with a history of A1 infection had higher titers throughout the study against A1 cells than domestic sheep < 1-yr-old and bighorns without a history of A1 infection. Both domestic and bighorn sheep had log2 titers of 8 to 12 against A2 cells and 6 to 12 against T10 cells during this time. Bighorn sheep in the VAC group had 2 to 32 fold titer increases for A1 cells by 2 wk post-vaccination (PV) compared to 0 to 2 fold increases in VAC domestic sheep. Two to 16 and 0 to 8 fold increases in antibodies titers to A2 and T10 cells, respectively, were detected in sera of both VAC groups. Sera of bighorn sheep with a history of respiratory disease and all domestic sheep had log2 leukotoxin neutralizing antibody titers of 4 to 14 in contrast to < or = 2 in sera of bighorn sheep without a history of respiratory disease. Neutralizing antibody titers of two bighorns without a history of respiratory disease in the VAC group increased from log2 0 to 5 in one and from 0 to 9 in the other 2 wk PV. Antibody increases in these animals were no longer evident at 16 wk PV while titers of animals with histories of disease remained relatively stable. The types and numbers of Pasteurella spp. isolated from nasal and pharyngeal swabs varied throughout the study without conclusive evidence of suppression of colonization. Although the animals were not experimentally challenged to determine the efficacy of the vaccine, one VAC and one NV bighorn sheep died following introduction of an A2 P. haemolytica strain when leukotoxin neutralizing antibodies had returned to pre-vaccination levels. This vaccine appeared to be safe for use in bighorn sheep and stimulated moderate but transient increases in antibody levels which should provide some protection against naturally occurring disease. A vaccine which would induce production of high and maintained antibodies against multiple strains of P. haemolytica would be valuable for use in bighorn sheep maintained in captivity or when captured for relocation.     

Experimental contact transmission of Pasteurella haemolytica from clinically normal domestic sheep causing pneumonia in Rocky Mountain bighorn sheep

Two Rocky Mountain bighorn lambs (Ovis canadensis canadensis) were held in captivity for 120 days before being housed with two domestic sheep. The lambs were clinically normal and had no Pasteurella spp. on nasal swab cultures. The domestic sheep were known to carry Pasteurella haemolytica biotype A in the nasal passages. After being in close contact for 19 days. P. haemolytica biotype A was cultured from nasal swabs of one of the bighorn lambs. By 26 days, both bighorn sheep developed coughs, were anorectic and became lethargic and nasal swabs yielded P. haemolytica biotype T, serotype 10. Twenty-nine days after contact, the lambs were necropsied and found to have extensive fibrinous bronchopneumonia. From affected tissues pure cultures of beta-hemolytic P. haemolytica biotype T, serotype 10 were grown. Both domestic sheep remained clinically normal and had no gross or microscopic lesions, but they carried the same P. haemolytica serotype in their tonsils. Behavioural observations gave no indication of stress in the bighorn lambs.     

Effect of anti-human pan-T monoclonal antibodies on lymphocyte proliferative and cytotoxic functions

Effects of anti-human pan-T-specific monoclonal antibodies of the Second International Workshop on Human Leucocyte Differentiation Antigens were investigated in a number of lymphocyte functional tests. Monoclonal antibodies blocking antibody-dependent cytotoxicity (ADCC), PWM-induced IL-2 release, or Con A- and PWM-induced lymphocyte proliferation were found among anti-CD2 and CD3 reagents. Inhibition of lectin-dependent cellular cytotoxicity (LDCC) was found as an exclusive effect of anti-CD2 (the sheep red cell receptor) antibodies. Several anti-CD2s blocked natural killer (NK) activity and/or PWM-induced interferon production. These two effects were exerted by antibodies against epitopes on resting T cells but not by those directed to activation epitopes. The inhibitory activity of individual antibodies in the LDCC and NK tests showed a good correlation. Also, PHA-mediated cytotoxicity (LDCC) and proliferation were in good correlation. Concerning anti-CD3 (T3) reagents, some effects were characteristic for the majority of the antibodies in this group. Namely, induction of proliferation, enhancement of IL-2-dependent cell division, IL-2 consumption by antibody-triggered cells, inhibition of mitogen-induced proliferation but not IL-2 and interferon production were observed. None of the CD3-specific reagents exerted all of these effects. In general, no correlation of the effects with immunoglobulin subclass or CD3 subcluster specificity could be found. Further epitope analysis and affinity data may be required to understand the basis of heterogeneity in functional effects of monoclonal antibodies to the CD3 molecule.     

Cleavage of the cell-surface O-sialoglycoproteins CD34, CD43, CD44, and CD45 by a novel glycoprotease from Pasteurella haemolytica.

The study of structural/functional characteristics of the cell-surface glycoproteins of leukocytes has led to a better understanding of the differentiation and maturation of hematopoietic cells. We have assessed the ability of a unique metalloprotease that is secreted by the bovine fibrinous pneumonia pathogen Pasteurella haemolytica, to cleave cell-surface glycoproteins expressed on human leukocytes. Biochemical analysis shows that the O-glycosylated cell surface Ag CD34, CD43 (leukosialin), CD44 (hyaluronic acid receptor), and CD45 (leukocyte common Ag), are all cleaved by this protease. Although these enzyme-sensitive structures contain N-linked glycans, they are all extensively glycosylated with O-linked carbohydrates, which are especially abundant on CD34 and CD43. In contrast, the glycoproteins CD18/11a,b,c (leukocyte integrins), CD71 (transferrin receptor), HLA class I, and 8A3 Ag, which contain N-linked glycans but no O-sialo-glycans, were resistant to the action of the enzyme. Inasmuch as previous studies using glycophorin A had indicated that the substrate specificity of this enzyme may be uniquely restricted to the cleavage of O-sialoglycoproteins, we have designated this activity, P. haemolytica glycoprotease. Immunofluorescence analysis with a variety of antibodies to different epitopes of the P. haemolytica glycoprotease-sensitive structures indicate that this enzyme may have widespread applications in epitope-mapping studies, and represents a novel tool with which to study structure/function relationships for O-sialoglycosylated cell-surface proteins. However, most significantly these results suggest that the P. haemolytica glycoprotease may be of use in the affinity purification and recovery of clinically important leukocyte subsets, such as primitive hematopoietic progenitors that express CD34.     

Cytokine-induced respiratory burst of human neutrophils: dependence on extracellular matrix proteins and CD11/CD18 integrins

Human polymorphonuclear leukocytes (PMN) released large quantities of hydrogen peroxide in response to tumor necrosis factor, but only when the cells were adherent to surfaces coated with extracellular matrix proteins. The PMN did not respond when exposed to cytokines and matrix proteins in suspension, or when exposed to cytokines while adherent to surfaces coated with stearic acid. PMN from children with genetic deficiency of the CD11/CD18 integrins underwent a normal respiratory burst upon adherence to uncoated polystyrene, but not in response to tumor necrosis factor when tested on polystyrene that was coated with serum, fibronectin, vitronectin, fibrinogen, thrombospondin, or laminin. Anti-CD18 antibodies, alone of sixteen antibodies tested, induced a similar defect in PMN from normal donors, when the PMN were tested on surfaces coated with serum, fibrinogen, thrombospondin, or laminin; no defect was induced by the anti-CD18 monoclonal antibody IB4 in normal PMN tested on surfaces coated with fibronectin or vitronectin. Thus, for cytokines to induce a respiratory burst in PMN, the cells must be able to use CD11/CD18 integrins and must interact with matrix proteins in the solid phase. CD11/CD18, which is already known to serve as a receptor for fibrinogen, may also be a receptor for thrombospondin and laminin. Finally, receptor(s) exist on PMN for fibronectin and vitronectin which are not blocked by the anti-CD18 antibody IB4 but which are nonetheless CD11/CD18 dependent.     

A bighorn sheep die-off in southern Colorado involving a Pasteurellaceae strain that may have originated from syntopic cattle

We investigated a pasteurellosis epizootic in free-ranging bighorn sheep (Ovis canadensis) wherein a Pasteurellaceae strain carried by syntopic cattle (Bos taurus) under severe winter conditions appeared to contribute to pneumonia in affected bighorns. Twenty-one moribund or dead bighorn sheep were found on the "Fossil Ridge" herd's winter range, Colorado, USA, between 13 December 2007 and 29 February 2008. Eight carcasses examined showed gross or microscopic evidence of acute to subacute fibrinous bronchopneumonia. All eight carcasses yielded at least one β-hemolytic Mannheimia haemolytica biogroup 1(±(G)) strain, and seven also yielded a β-hemolytic Bibersteinia trehalosi biogroup 4 (CDS) strain; evidence of Pasteurella multocida, Mycoplasma ovipneumoniae, and parainfluenza 3 and bovine respiratory syncytial viruses was also detected. Isolates of β-hemolytic Manneimia haemolytica biogroup 1(G) from a bighorn carcass and a syntopic cow showed 99.5% similarity in genetic fingerprints; B. trehalosi biogroup 4(CDS) isolates were ≥94.9% similar to an isolate from a nearby bighorn herd. Field and laboratory observations suggested that pneumonia in affected bighorns may have been caused by a combination of pathogens including two pathogenic Pasteurellaceae strains--one likely of cattle origin and one likely of bighorn origin--with infections in some cases perhaps exacerbated by other respiratory pathogens and severe weather conditions. Our and others' findings suggest that intimate interactions between wild sheep and cattle should be discouraged as part of a comprehensive approach to health management and conservation of North American wild sheep species.     

Involvement of beta 2-integrins in the migration of human natural killer cells.

Human large granular lymphocytes with the NK cell phenotype (CD16+ or CD56+CD3-) were greatly enriched among the cells which migrated spontaneously through untreated or albumin-coated, 3-microns pore size polycarbonate filters for 1 to 8 h. Three days of rIL-2 treatment (300 IU/ml) and 3 to 5 wk of rIL-2 treatment (100 IU/ml) generated a 2.7 +/- 0.9-fold and 5.6 +/- 0.8-fold increase in cell migration, respectively. The adhesion and subsequent migration of freshly isolated NK cells was mainly mediated by CD11b/CD18, because migration could be inhibited by 80 +/- 8% anti-CD11b (Mac-1) antibodies but not with antibodies against CD11a (LFA-1) or CD11c (p150,95), the other alpha-chains of the beta 2-integrins. After rIL-2 activation, however, CD11a/CD18 was the major receptor utilized in migration, inasmuch as anti-CD11a antibody caused a 69 +/- 8% reduction in the number of migrated cells. Anti-CD11b antibody decreased migration by 43 +/- 12%, and together these antibodies inhibited migration by 82 +/- 7%. Anti-CD11a alone did not have any effect on adhesion, but CD11a/CD18 cooperated in the adhesion because anti-CD11b decreased adhesion by 40 +/- 11% and together these antibodies inhibited adhesion by 74 +/- 6%. The ability of large granular lymphocytes to rapidly utilize beta 2-integrins and unidentified ubiquitous ligands for binding and migration may be significant for their capacity to function in the first line of immune defense under highly variable conditions.     

Role of the CD45 (T-200) molecule in anti-CD3-triggered T cell-mediated cytotoxicity

NK-depleted human peripheral blood lymphocytes can be modulated with anti-CD3 to kill certain targets during 3-hr cytotoxicity assays. When triggered by anti-CD3 antibody, these effector T cells killed only NK-sensitive targets, such as K562 and HEL 92.1.7, and NK-resistant targets, such as Daudi, whose killing is inhibited by anti-CD45 (T-200) monoclonal antibodies, such as 13.3. NK-sensitive targets, MOLT-4, U266/AF10, Jurkat, and CCFR-CEM, and 10 NK-resistant cell lines, including Raji, IM-9, U698, U937, and GM-1056, whose killing is not inhibited by anti-CD45 monoclonal antibodies, were not killed by alpha-CD3-T effectors, suggesting that the CD45 molecule may be involved in the killing process. Anti-CD3-triggered T cell killing of target cells was inhibited greater than 95% by the monoclonal antibody 13.3. This inhibition of cytotoxicity by 13.3 was not due to competition of this IgG1 antibody for Fc receptor binding site on the target cell, since the IgG1 monoclonal antibody anti-beta 2-microglobulin did not block cytotoxicity. Single cell assays and calcium pulse assays showed that CD45 is involved in a postbinding, pre-calcium-dependent stage, similar to that shown for NK cytotoxicity. There was a relative shift of importance of different epitopes of CD45 in anti-CD3-T cytotoxicity compared to NK cytotoxicity. Anti-CD45 antibodies which bind to the C terminus end of the molecule played a more important role in anti-CD3-T cytotoxicity than NK cytotoxicity. Thus, a subset of T cells exists that exhibits anti-CD3-triggered non-MHC-restricted killing of certain NK-sensitive and NK-resistant targets in association with a CD45 molecule which is functionally different from the NK CD45 molecule.     

Role of CD2 and IL 1 beta in T cell responsiveness to IL 2

We have evaluated the effects of an anti-CD2 monoclonal antibody (moAb), Leu-5b, on inhibition of lymphocyte responsiveness. Given previous reports that anti-CD2 moAb prevent production of interleukin 2 (IL 2), as well as up-regulation of IL 2 receptor, we chose a test system in which lymphocytes were activated by exogenously added recombinant IL 2 (rIL 2). Our studies thus allowed us to evaluate other possible mechanisms by which anti-CD2 moAb could act. We found that Leu-5b not only blocked the proliferative response of human peripheral blood leukocytes (PBL) to IL 2, but also the development of cytotoxicity as assayed in lectin-dependent cytotoxicity (LDCC). Addition of recombinant interleukin 1 beta (rIL 1 beta) reconstituted the proliferative response in the presence of Leu-5b but did not allow development of cytotoxicity. Our findings are consistent with the concept that Leu-5b delivers a signal to IL 2-responsive cells that prevents their subsequent proliferation and development of cytotoxicity, but that only the former event, i.e., proliferation, is reconstituted by the addition of IL 1 beta.     

Epidemic pasteurellosis in a bighorn sheep population coinciding with the appearance of a domestic sheep

A pneumonia epidemic reduced bighorn sheep (Ovis canadensis) survival and recruitment during 1997-2000 in a population comprised of three interconnected wintering herds (Kenosha Mountains, Sugarloaf Mountain, Twin Eagles) that inhabited the Kenosha and Tarryall Mountain ranges in central Colorado, USA. The onset of this epidemic coincided temporally and spatially with the appearance of a single domestic sheep (Ovis aires) on the Sugarloaf Mountain herd's winter range in December 1997. Although only bighorns in the Sugarloaf Mountain herd were affected in 1997-98, cases also occurred during 1998-99 in the other two wintering herds, likely after the epidemic spread via established seasonal movements of male bighorns. In all, we located 86 bighorn carcasses during 1997-2000. Three species of Pasteurella were isolated in various combinations from affected lung tissues from 20 bighorn carcasses where tissues were available and suitable for diagnostic evaluation; with one exception, beta-hemolytic mannheimia (Pasteurella) haemolytica (primarily reported as biogroup 1(G) or 1(alphaG)) was isolated from lung tissues of cases evaluated during winter 1997-98. The epidemic dramatically lowered adult bighorn monthly survival in all three herds; a model that included an acute epidemic effect, differing between sexes and with vaccination status, that diminished linearly over the next 12 mo best represented field data. In addition to the direct mortality associated with epidemics in these three herds, lamb recruitment in years following the pneumonia epidemic also was depressed as compared to years prior to the epidemic. Based on observations presented here, pasteurellosis epidemics in free-ranging bighorn sheep can arise through incursion of domestic sheep onto native ranges, and thus minimizing contact between domestic and bighorn sheep appears to be a logical principle for bighorn sheep conservation.     

Expression of CD30 ligand (CD153) on murine activated T cells

CD30, a member of the TNF receptor family, has been implicated in the activation of T cells and B cells. In the present study, we characterized the expression and function of murine CD30 ligand (mCD153) by utilizing mCD153 transfectants and a novel mAb against mCD153 (RM153), which can inhibit the binding of murine CD30 to mCD153. The mCD153 transfectants did not co-stimulate the proliferation of anti-CD3-stimulated naive T cells but enhanced the proliferation of anti-CD28-co-stimulated T cells. The mCD153 transfectants exhibited a potent co-stimulatory activity for proliferation of pre-activated T cells that expressed CD30 after anti-CD3 and anti-CD28 stimulation. In contrast to the CD30 expression on naive T cells that required anti-CD28 co-stimulation, mCD153 expression was observed on anti-CD3-stimulated T cells without the anti-CD28 co-stimulation, predominantly on CD4(+) T cells with a transient kinetics which peaked at 24 h but disappeared at 48 h. In contrast to the preferential expression of CD30 on Th2 cells, mCD153 was expressed on both Th1 and Th2 cells after anti-CD3 stimulation. These results indicated a differential regulation of CD30 and CD153 expression in T cells, which may be relevant to immuno-regulatory role of the CD30-CD153 interaction.     

Using ribosomal RNA gene restriction patterns in distinguishing isolates of Pasteurella haemolytica from bighorn sheep (Ovis canadensis).

Pasteurella haemolytica isolates (n = 31) from two isolated captive herds of Rocky Mountain bighorn sheep (Ovis canadensis canadensis) were characterized and compared phenotypically (biotype, serotype, hemolytic activity) and by a genomic fingerprinting method known as ribotyping. Seven to nine distinct phenotypes were observed. Depending on the method used for serotyping, one to three phenotypes were common to both herds. Eighteen isolates, recovered from both herds, were non-hemolytic, biotype T, indirect hemagglutination assay serotype 4. Ribotyping, a method for highlighting genetically conserved deoxyribonucleic acid restriction site heterogeneity with a 32P-labelled Escherichia coli ribosomal ribonucleic acid probe, produced six to eight distinct ribotype pattern groups within the 31 P. haemolytica isolates, depending on the restriction enzyme used. In contrast to phenotypes, ribotypes appeared unique to each herd, and ribotyping helped to further differentiate some isolates of the same biotype and serotype. In addition, ribotyping provided an alternative means for evaluating relationships between isolates differing in hemolytic activity but which were otherwise phenotypically identical. We propose that ribotyping may be a useful adjunct to other bacterial characterization methods in studying the epizootiology of pasteurellosis in bighorn sheep.     

CD27-mediated activation of murine NK cells

CD27, a member of the TNF receptor superfamily, has been implicated in T cell activation, T cell development, and T cell-dependent Ab production by B cells. In the present study we examined the expression and function of CD27 on murine NK cells. Murine NK cells constitutively expressed CD27 on their surface. Stimulation with immobilized anti-CD27 mAb or murine CD27 ligand (CD70) transfectans solely could induce proliferation and IFN-gamma production of freshly isolated NK cells and enhanced the proliferation and IFN-gamma production of anti-NK1.1-sutimulated NK cells. Although NK cell cytotoxicity was not triggered by anti-CD27 mAb or against CD70 transfectants, prestimulation via CD27 enhanced the cytotoxic activity of NK cells in an IFN-gamma-dependent manner. These results suggest that CD27-mediated activation may be involved in the NK cell-mediated innate immunity against virus-infected or transformed cells expressing CD70.     

Proximity-Dependent Inhibition of Growth of Mannheimia haemolytica by Pasteurella multocida

Mannheimia haemolytica, Pasteurella multocida, and Bibersteinia trehalosi have been identified in the lungs of pneumonic bighorn sheep (BHS; Ovis canadensis). Of these pathogens, M. haemolytica has been shown to consistently cause fatal pneumonia in BHS under experimental conditions. However, M. haemolytica has been isolated by culture less frequently than the other bacteria. We hypothesized that the growth of M. haemolytica is inhibited by other bacteria in the lungs of BHS. The objective of this study was to determine whether P. multocida inhibits the growth of M. haemolytica. Although in monoculture both bacteria exhibited similar growth characteristics, in coculture with P. multocida there was a clear inhibition of growth of M. haemolytica. The inhibition was detected at mid-log phase and continued through the stationary phase. When cultured in the same medium, the growth of M. haemolytica was inhibited when both bacteria were separated by a membrane that allowed contact (pore size, 8.0 μm) but not when they were separated by a membrane that limited contact (pore size, 0.4 μm). Lytic bacteriophages or bactericidal compounds could not be detected in the culture supernatant fluid from monocultures of P. multocida or from P. multocida-M. haemolytica cocultures. These results indicate that P. multocida inhibits the growth of M. haemolytica by a contact- or proximity-dependent mechanism. If the inhibition of growth of M. haemolytica by P. multocida occurs in vivo as well, it could explain the inconsistent isolation of M. haemolytica from the lungs of pneumonic BHS.     

Genetic cause of leukocyte adhesion molecule deficiency. Abnormal splicing and a missense mutation in a conserved region of CD18 impair cell surface expression of beta 2 integrins.

Patients with leukocyte adhesion molecule (CD11/CD18, beta 2 integrins) deficiency have structural defects in the common beta subunit (CD18), which prevent heterodimer formation and normal cell surface expression of these receptors, leading to life-threatening bacterial infections. To elucidate the nature of these defects in a patient with partial (type II) deficiency, abnormal CD18 cDNA clones were isolated, using the polymerase chain reaction to amplify the patient's B cell-derived cDNAs. Sequence analysis revealed two mutant alleles. cDNA clones, representing a maternal allele, contained both a 12-base pair insertion resulting in an in-frame addition of four amino acids between P247 and E248 and a C1756----T nucleotide transition, resulting in an R586----W substitution in the normal CD18 protein. The inframe insertion arose by a single nucleotide C----A transversion in the 3' terminus of an intron, generating aberrant splice acceptor site. Other cDNA clones contained an A1052----G nucleotide transition not present in either parent which resulted in an N351----S substitution. To determine the functional importance of these changes, cDNA encoding a normal alpha chain (CD11b) was cotransfected into COS with CD18 cDNAs encoding for wild-type, maternal mutant allele, or CD18 containing N351----S substitution. Immunostaining of transfectants with anti-CD18 monoclonal antibodies revealed no cell surface expression of the maternal mutant CD18, and 22% surface expression of N351----S CD18. Both the insertion and the N351----S mutations occurred in a 250 amino acid extracellular region of CD18 that is highly conserved among beta integrins supporting a role for this region in heterodimer formation.