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Robert L. Davies - One of the best experts on this subject based on the ideXlab platform.
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Diversity of temperate bacteriophages induced in bovine and ovine Mannheimia haemolytica isolates and identification of a new P2-like phage
FEMS microbiology letters, 2006Co-Authors: Robert L. Davies, Inkyoung LeeAbstract:The diversity of temperate bacteriophages was examined in 32 Mannheimia haemolytica, six Mannheimia glucosida and four Pasteurella trehalosi isolates. Phage particles were induced and identified by electron microscopy in 24 (75%) M. haemolytica isolates, but in only one (17%) M. glucosida and one (25%) P. trehalosi isolate. The M. haemolytica phages were relatively diverse as seven Siphoviridae, 15 Myoviridae and two Podoviridae-like phages were identified; the Myoviridae-type phages also exhibited structural variation of their tails. The bacteriophages induced in M. glucosida and P. trehalosi were of the Myoviridae type. Restriction endonuclease (RE) analysis identified nine distinct RE types among the M. haemolytica bacteriophages, providing further evidence of their relative diversity. A limited number of phages caused plaques on indicator strains and the phages exhibited a narrow host range. A subgroup of 11 bovine serotype A1 and A6 isolates contained Myoviridae-type phages of the same RE type (type A), but these differed in their abilities to infect and form plaques on the same panel of indicator strains. A P2-like phage (φPHL213.1), representative of the RE type A phages, was identified from the incomplete M. haemolytica genome sequence. The φPHL213.1 genome contains previously unidentified genes and represents a new member of the P2 phage family.
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sequence diversity and molecular evolution of the heat modifiable outer membrane protein gene ompa of mannheimia Pasteurella haemolytica mannheimia glucosida and Pasteurella trehalosi
Journal of Bacteriology, 2004Co-Authors: Robert L. DaviesAbstract:The OmpA (or heat-modifiable) protein is a major structural component of the outer membranes of gram-negative bacteria. The protein contains eight membrane-traversing β-strands and four surface-exposed loops. The genetic diversity and molecular evolution of OmpA were investigated in 31 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains by comparative nucleotide sequence analysis. The OmpA proteins of M. haemolytica and M. glucosida contain four hypervariable domains located at the distal ends of the surface-exposed loops. The hypervariable domains of OmpA proteins from bovine and ovine M. haemolytica isolates are very different but are highly conserved among strains from each of these two host species. Fourteen different alleles representing four distinct phylogenetic classes, classes I to IV, were identified in M. haemolytica and M. glucosida. Class I, II, and IV alleles were associated with bovine M. haemolytica, ovine M. haemolytica, and M. glucosida strains, respectively, whereas class III alleles were present in certain M. haemolytica and M. glucosida isolates. Class I and II alleles were associated with divergent lineages of bovine and ovine M. haemolytica strains, respectively, indicating a history of horizontal DNA transfer and assortative (entire gene) recombination. Class III alleles have mosaic structures and were derived by horizontal DNA transfer and intragenic recombination. Our findings suggest that OmpA is under strong selective pressure from the host species and that it plays an important role in host adaptation. It is proposed that the OmpA protein of M. haemolytica acts as a ligand and is involved in binding to specific host cell receptor molecules in cattle and sheep. P. trehalosi expresses two OmpA homologs that are encoded by different tandemly arranged ompA genes. The P. trehalosi ompA genes are highly diverged from those of M. haemolytica and M. glucosida, and evidence is presented to suggest that at least one of these genes was acquired by horizontal DNA transfer.
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sequence diversity and molecular evolution of the heat modifiable outer membrane protein gene ompa of mannheimia Pasteurella haemolytica mannheimia glucosida and Pasteurella trehalosi
Journal of Bacteriology, 2004Co-Authors: Robert L. Davies, Inkyoung LeeAbstract:The OmpA (or heat-modifiable) protein is a major structural component of the outer membranes of gram-negative bacteria. The protein contains eight membrane-traversing beta-strands and four surface-exposed loops. The genetic diversity and molecular evolution of OmpA were investigated in 31 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains by comparative nucleotide sequence analysis. The OmpA proteins of M. haemolytica and M. glucosida contain four hypervariable domains located at the distal ends of the surface-exposed loops. The hypervariable domains of OmpA proteins from bovine and ovine M. haemolytica isolates are very different but are highly conserved among strains from each of these two host species. Fourteen different alleles representing four distinct phylogenetic classes, classes I to IV, were identified in M. haemolytica and M. glucosida. Class I, II, and IV alleles were associated with bovine M. haemolytica, ovine M. haemolytica, and M. glucosida strains, respectively, whereas class III alleles were present in certain M. haemolytica and M. glucosida isolates. Class I and II alleles were associated with divergent lineages of bovine and ovine M. haemolytica strains, respectively, indicating a history of horizontal DNA transfer and assortative (entire gene) recombination. Class III alleles have mosaic structures and were derived by horizontal DNA transfer and intragenic recombination. Our findings suggest that OmpA is under strong selective pressure from the host species and that it plays an important role in host adaptation. It is proposed that the OmpA protein of M. haemolytica acts as a ligand and is involved in binding to specific host cell receptor molecules in cattle and sheep. P. trehalosi expresses two OmpA homologs that are encoded by different tandemly arranged ompA genes. The P. trehalosi ompA genes are highly diverged from those of M. haemolytica and M. glucosida, and evidence is presented to suggest that at least one of these genes was acquired by horizontal DNA transfer.
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mosaic structure and molecular evolution of the leukotoxin operon lktcabd in mannheimia Pasteurella haemolytica mannheimia glucosida and Pasteurella trehalosi
Journal of Bacteriology, 2002Co-Authors: Robert L. Davies, Susan Campbell, Thomas S WhittamAbstract:The mosaic structure and molecular evolution of the leukotoxin operon (lktCABD) was investigated by nucleotide sequence comparison of the lktC, lktB, and lktD genes in 23 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains. Sequence variation in the lktA gene has been described previously (R. L. Davies et al., J. Bacteriol. 183:1394–1404, 2001). The leukotoxin operon of M. haemolytica has a complex mosaic structure and has been derived by extensive inter- and intraspecies horizontal DNA transfer and intragenic recombination events. However, the pattern of recombination varies throughout the operon and among the different evolutionary lineages of M. haemolytica. The lktA and lktB genes have the most complex mosaic structures with segments derived from up to four different sources, including M. glucosida and P. trehalosi. In contrast, the lktD gene is highly conserved in M. haemolytica. The lktC, lktA, and lktB genes of strains representing the major ovine lineages contain recombinant segments derived from bovine or bovine-like serotype A2 strains. These findings support the previous conclusion that host switching of bovine A2 strains from cattle to sheep has played a major role in the evolution of the leukotoxin operon in ovine strains of M. haemolytica. Homologous segments of donor and recipient alleles are identical, or nearly identical, indicating that the recombinational exchanges occurred relatively recent in evolutionary terms. The 5′ and 3′ ends of the operon are highly conserved in M. haemolytica, which suggests that multiple horizontal exchanges of the complete operon have occurred by a common mechanism such as transduction. Although the lktA and lktB genes both have complex mosaic structures and high nucleotide substitution rates, the amino acid diversity of LktB is significantly lower than that of LktA due to a higher degree of evolutionary constraint against amino acid replacement. The recombinational exchanges within the leukotoxin operon have had greatest effect on LktA and probably provide an adaptive advantage against the host antibody response by generating novel antigenic variation at surface-exposed sites.
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sequence diversity and molecular evolution of the leukotoxin lkta gene in bovine and ovine strains of mannheimia Pasteurella haemolytica
Journal of Bacteriology, 2001Co-Authors: Robert L. Davies, Thomas S Whittam, R K SelanderAbstract:The molecular evolution of the leukotoxin structural gene (lktA) of Mannheimia (Pasteurella) haemolytica was investigated by nucleotide sequence comparison of lktA in 31 bovine and ovine strains representing the various evolutionary lineages and serotypes of the species. Eight major allelic variants (1.4 to 15.7% nucleotide divergence) were identified; these have mosaic structures of varying degrees of complexity reflecting a history of horizontal gene transfer and extensive intragenic recombination. The presence of identical alleles in strains of different genetic backgrounds suggests that assortative (entire gene) recombination has also contributed to strain diversification in M. haemolytica. Five allelic variants occur only in ovine strains and consist of recombinant segments derived from as many as four different sources. Four of these alleles consist of DNA (52.8 to 96.7%) derived from the lktA gene of the two related species Mannheimia glucosida and Pasteurella trehalosi, and four contain recombinant segments derived from an allele that is associated exclusively with bovine or bovine-like serotype A2 strains. The two major lineages of ovine serotype A2 strains possess lktA alleles that have very different evolutionary histories and encode divergent leukotoxins (5.3% amino acid divergence), but both contain segments derived from the bovine allele. Homologous segments of donor and recipient alleles are identical or nearly identical, indicating that the recombination events are relatively recent and probably postdate the domestication of cattle and sheep. Our findings suggest that host switching of bovine strains from cattle to sheep, together with inter- and intraspecies recombinational exchanges, has played an important role in generating leukotoxin diversity in ovine strains. In contrast, there is limited allelic diversity of lktA in bovine strains, suggesting that transmission of strains from sheep to cattle has been less important in leukotoxin evolution.
Inkyoung Lee - One of the best experts on this subject based on the ideXlab platform.
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Diversity of temperate bacteriophages induced in bovine and ovine Mannheimia haemolytica isolates and identification of a new P2-like phage
FEMS microbiology letters, 2006Co-Authors: Robert L. Davies, Inkyoung LeeAbstract:The diversity of temperate bacteriophages was examined in 32 Mannheimia haemolytica, six Mannheimia glucosida and four Pasteurella trehalosi isolates. Phage particles were induced and identified by electron microscopy in 24 (75%) M. haemolytica isolates, but in only one (17%) M. glucosida and one (25%) P. trehalosi isolate. The M. haemolytica phages were relatively diverse as seven Siphoviridae, 15 Myoviridae and two Podoviridae-like phages were identified; the Myoviridae-type phages also exhibited structural variation of their tails. The bacteriophages induced in M. glucosida and P. trehalosi were of the Myoviridae type. Restriction endonuclease (RE) analysis identified nine distinct RE types among the M. haemolytica bacteriophages, providing further evidence of their relative diversity. A limited number of phages caused plaques on indicator strains and the phages exhibited a narrow host range. A subgroup of 11 bovine serotype A1 and A6 isolates contained Myoviridae-type phages of the same RE type (type A), but these differed in their abilities to infect and form plaques on the same panel of indicator strains. A P2-like phage (φPHL213.1), representative of the RE type A phages, was identified from the incomplete M. haemolytica genome sequence. The φPHL213.1 genome contains previously unidentified genes and represents a new member of the P2 phage family.
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molecular evolution of mannheimia Pasteurella haemolytica mannheimia glucosida and Pasteurella trehalosi and characterization of temperate bacteriophages
2005Co-Authors: Inkyoung LeeAbstract:Comparative nucleotide sequence analysis of nineteen genes of different functional classes was carried out in representative isolates of Mannheimia haemolytica (10 to 32 isolates), Mannheimia glucosida (1 to 6 isolates), and Pasteurella trehalosi (1 to 4 isolates). The genes include one DNA repair and recombination gene (recA), nine metabolic enzyme genes aroA, asd, galE, gap, gnd, g6pd, mdh, mtlD, and pmm), one secreted protein gene (gcp), four periplasm-associated lipoprotein genes (plpA, plpB, plpC, and plpD), and four outer membrane protein genes (ompA, tbpB, tbpA, and wza). This study was inspired by previous work which had revealed that recombination involving M. glucosida and P. trehalosi, together with host switching of isolates from cattle to sheep, have played important roles in the evolution of the M. haemolytica leukotoxin operon (Davies et al., 2001; 2002). Comparative nucleotide sequence analysis of the nineteen genes indicated that only single nucleotide substitutions had occurred in the recA, asd, gnd, g6pd, mtlD, and gcp genes of M haemolytica. A phylogenetic tree based on their concatenated sequences supported the evolutionary relationships of isolates of M. haemolytica based on previous MLEE studies. In contrast, the aroA, gap, mdh, pmm, plpA, plpB, plpC, plpD, ompA, tbpB, tbpA, and wza genes showed evidence of varying degrees of mosaic structure which suggests that horizontal DNA transfer and intragenic recombination have played important roles in the evolution of these genes. Recombinant segments of M haemolytica genes have been derived from M glucosida and other unidentified sources indicating that DNA from other bacterial species has contributed to the evolution of M haemolytica. Extensive recombinant exchanges have occurred in the outer membrane protein genes ompA, tbpB, tbpA, and wza. The different tree topologies and different patterns of nucleotide and amino acid diversity of these four genes indicate that they have different evolutionary histories. However, the evolutionary histories of these four genes support the previously proposed view that M. haemolytica diversity has been influenced by the transmission of isolates from cattle to sheep and vice versa, which is probably linked to the domestication of these species. The temperate bacteriophages of representative isolates of M. haemolytica, M. glucosida, and P. trehalosi were also investigated in this study since phages are known to mediate horizontal DNA transfer. Induction of bacteriophages with mitomycin C and examination by electron microscopy revealed that temperate phages were more prevalent in M. haemolytica (24 of 32 isolates) than M. glucosida (one of six isolates) and P. trehalosi (one of four isolates). Genetic variation of the phages was assessed by restriction endonuclease analysis and host range was determined by plague assay. Phage DNA was successfully isolated from 15 M. haemolytica, one M glucosida, and one P. trehalosi isolates and restriction endonuclease analysis identified nine different RE types (A to I) in M. haemolytica, one RE type (J) in M. glucosida, and one RE type (K) in P. trehalosi. Plaque assay revealed that 13 M. haemolytica, one M. glucosida, and one P. trehalosi lysates could produce lysis zones on one to six M. haemolytica, two M. glucosida, and one P. trehalosi indicator isolates, respectively. Using restriction endonuclease analysis and genomic analysis of the M. haemolytica genome sequence, phages of RE type A were shown to be very similar to P2 phages of E. coli. The annotated genome of this phage type was derived from the bacterial unannotated genomic sequence and compared with the genome of P2 phages.
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sequence diversity and molecular evolution of the heat modifiable outer membrane protein gene ompa of mannheimia Pasteurella haemolytica mannheimia glucosida and Pasteurella trehalosi
Journal of Bacteriology, 2004Co-Authors: Robert L. Davies, Inkyoung LeeAbstract:The OmpA (or heat-modifiable) protein is a major structural component of the outer membranes of gram-negative bacteria. The protein contains eight membrane-traversing beta-strands and four surface-exposed loops. The genetic diversity and molecular evolution of OmpA were investigated in 31 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains by comparative nucleotide sequence analysis. The OmpA proteins of M. haemolytica and M. glucosida contain four hypervariable domains located at the distal ends of the surface-exposed loops. The hypervariable domains of OmpA proteins from bovine and ovine M. haemolytica isolates are very different but are highly conserved among strains from each of these two host species. Fourteen different alleles representing four distinct phylogenetic classes, classes I to IV, were identified in M. haemolytica and M. glucosida. Class I, II, and IV alleles were associated with bovine M. haemolytica, ovine M. haemolytica, and M. glucosida strains, respectively, whereas class III alleles were present in certain M. haemolytica and M. glucosida isolates. Class I and II alleles were associated with divergent lineages of bovine and ovine M. haemolytica strains, respectively, indicating a history of horizontal DNA transfer and assortative (entire gene) recombination. Class III alleles have mosaic structures and were derived by horizontal DNA transfer and intragenic recombination. Our findings suggest that OmpA is under strong selective pressure from the host species and that it plays an important role in host adaptation. It is proposed that the OmpA protein of M. haemolytica acts as a ligand and is involved in binding to specific host cell receptor molecules in cattle and sheep. P. trehalosi expresses two OmpA homologs that are encoded by different tandemly arranged ompA genes. The P. trehalosi ompA genes are highly diverged from those of M. haemolytica and M. glucosida, and evidence is presented to suggest that at least one of these genes was acquired by horizontal DNA transfer.
Michael W. Miller - One of the best experts on this subject based on the ideXlab platform.
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novel protease produced by a Pasteurella trehalosi serotype 10 isolate from a pneumonic bighorn sheep characteristics and potential relevance to protection
Veterinary Microbiology, 2003Co-Authors: Heather J Mcneil, Jennifer A. Conlon, Patricia E Shewen, Michael W. MillerAbstract:Abstract A strain of Pasteurella trehalosi serotype 10, E CO -100, isolated from a bighorn sheep that had succumbed to pneumonic pasteurellosis during an epizootic, was compared to well-characterized strains of P. trehalosi serotype 10 and Mannheimia haemolytica serotype 1. The gene for leukotoxin A ( lktA ) from E CO -100 was sequenced and found to be identical on an amino acid basis to a published sequence for lktA from P. trehalosi serotype 10. However, the toxic activity in culture supernatant measured over time for E CO -100 was quite different from reference strains. Typically, the ability of the supernatant to lyse target cells increases over time corresponding to the logarithmic growth of the organism, peaks at mid to late phase, then declines gradually. Supernatant from E CO -100 exhibited a sharp decline in toxicity after mid-logarithmic growth to undetectable levels. Investigation of this anomaly using a commercial kit with a porcine gelatin/bovine albumin substrate matrix revealed high protease activity in the supernatant of this strain compared to another P. trehalosi serotype 10 and to a M. haemolytica serotype 1. Protease activity was also visualized using gelatin based zymogram gels. This protease was not substrate specific as it was shown to degrade leukotoxin. Activity was neutralized by bighorn sera in a titratable manner. There was an association between the ability to neutralize protease and low pneumonic lung scores in bighorn sheep experimentally challenged with E CO -100 ( r =0.5, P =0.1). This previously unidentified protease may be an important protective antigen in vaccines designed to prevent pneumonic pasteurellosis resulting from P. trehalosi in bighorn sheep.
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Mannheimia haemolytica serotype 1 and Pasteurella trehalosi serotype 10 culture supernatants contain fibrinogen-binding proteins.
Veterinary Immunology and Immunopathology, 2002Co-Authors: Heather J Mcneil, Reggie Y.c. Lo, Jennifer A. Conlon, Patricia E Shewen, Michael W. MillerAbstract:Abstract Fibrinogen-binding proteins were found in the culture supernatants of Mannheimia haemolytica serotype 1 (ATCC 43270) and Pasteurella trehalosi serotype 10 (ECO-100). Sheep fibrinogen was biotinylated and shown to bind to proteins in the culture supernatants by modified western blot. Fibrinogen-binding proteins in the culture supernatant may be important virulence factors leading to the characteristic fibrinous pneumonia caused by these organisms and may be critical antigenic targets for immune prophylaxis.
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evaluation of ewe vaccination as a tool for increasing bighorn lamb survival following pasteurellosis epizootics
Journal of Wildlife Diseases, 2001Co-Authors: Frances E Cassirer, Karen M Rudolph, Pat Fowler, Victor L Coggins, David L Hunter, Michael W. MillerAbstract:We conducted field and laboratory experiments to evaluate whether treating pregnant bighorn ewes with a combination of an experimental Pasteurella trehalosi and Mannheimia haemolytica (formerly P. haemolytica) vaccine and a commercially-available bovine P. multocida and M. haemolytica vaccine would increase lamb survival following a pneumonia epidemic. Three free-ranging bighorn herds affected by pasteurellosis outbreaks between November 1995 and June 1996 were included in the field experiment. Post-epidemic lamb survival was low in all three herds in 1996, with November lamb:ewe ratios of ≤8:100. In March 1997, thirty-six ewes (12/herd) were captured and radiocollared. Half of the ewes captured in each herd were randomly selected to receive both vaccines; the other half were injected with 0.9% saline solution as controls. Lambs born to radiocollared ewes were observed two or more times per week and were considered to have survived if they were alive in October 1997, about 6 mo after birth. Lamb survival ...
Schwarz Stefan - One of the best experts on this subject based on the ideXlab platform.
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Plasmid-mediated florfenicol resistance in Pasteurella trehalosi.
Journal of Antimicrobial Chemotherapy, 2006Co-Authors: Corinna Kehrenberg, Danièle Meunier, Hayette Targant, Axel Cloeckaert, Jean-yves Madec, Schwarz StefanAbstract:OBJECTIVES: A florfenicol-resistant Pasteurella trehalosi isolate from a calf was investigated for the presence and the location of the gene floR. METHODS: The P. trehalosi isolate 13698 was investigated for its in vitro susceptibility to antimicrobial agents and its plasmid content. A 14.9 kb plasmid, designated pCCK13698, was identified by transformation into Pasteurella multocida to mediate resistance to florfenicol, chloramphenicol and sulphonamides. The plasmid was sequenced completely and analysed for its structure and organization. RESULTS: Plasmid pCCK13698 exhibited extended similarity to plasmid pHS-Rec from Haemophilus parasuis including the region carrying the parA, repB, rec and int genes. Moreover, it revealed similarities to plasmid RSF1010 in the parts covering the mobC and repA-repC genes and to plasmid pMVSCS1 in the parts covering the sul2-catA3-strA gene cluster. Moreover, the floR gene area corresponded to that of transposon TnfloR. In addition, two complete insertion sequences were detected that were highly similar to IS1593 from Mannheimia haemolytica and IS26 from Enterobacteriaceae. Several potential recombination sites were identified that might explain the development of plasmid pCCK13698 by recombination events. CONCLUSIONS: The results of this study showed that in the bovine pathogen P. trehalosi, floR-mediated resistance to chloramphenicol and florfenicol was associated with a plasmid, which also carried functionally active genes for resistance to sulphonamides (sul2) and chloramphenicol (catA3). This is to the best of our knowledge the first report of resistance genes in P. trehalosi and only the second report of the presence of a florfenicol-resistance gene in target bacteria of the family Pasteurellaceae.
Heather J Mcneil - One of the best experts on this subject based on the ideXlab platform.
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novel protease produced by a Pasteurella trehalosi serotype 10 isolate from a pneumonic bighorn sheep characteristics and potential relevance to protection
Veterinary Microbiology, 2003Co-Authors: Heather J Mcneil, Jennifer A. Conlon, Patricia E Shewen, Michael W. MillerAbstract:Abstract A strain of Pasteurella trehalosi serotype 10, E CO -100, isolated from a bighorn sheep that had succumbed to pneumonic pasteurellosis during an epizootic, was compared to well-characterized strains of P. trehalosi serotype 10 and Mannheimia haemolytica serotype 1. The gene for leukotoxin A ( lktA ) from E CO -100 was sequenced and found to be identical on an amino acid basis to a published sequence for lktA from P. trehalosi serotype 10. However, the toxic activity in culture supernatant measured over time for E CO -100 was quite different from reference strains. Typically, the ability of the supernatant to lyse target cells increases over time corresponding to the logarithmic growth of the organism, peaks at mid to late phase, then declines gradually. Supernatant from E CO -100 exhibited a sharp decline in toxicity after mid-logarithmic growth to undetectable levels. Investigation of this anomaly using a commercial kit with a porcine gelatin/bovine albumin substrate matrix revealed high protease activity in the supernatant of this strain compared to another P. trehalosi serotype 10 and to a M. haemolytica serotype 1. Protease activity was also visualized using gelatin based zymogram gels. This protease was not substrate specific as it was shown to degrade leukotoxin. Activity was neutralized by bighorn sera in a titratable manner. There was an association between the ability to neutralize protease and low pneumonic lung scores in bighorn sheep experimentally challenged with E CO -100 ( r =0.5, P =0.1). This previously unidentified protease may be an important protective antigen in vaccines designed to prevent pneumonic pasteurellosis resulting from P. trehalosi in bighorn sheep.
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Mannheimia haemolytica serotype 1 and Pasteurella trehalosi serotype 10 culture supernatants contain fibrinogen-binding proteins.
Veterinary Immunology and Immunopathology, 2002Co-Authors: Heather J Mcneil, Reggie Y.c. Lo, Jennifer A. Conlon, Patricia E Shewen, Michael W. MillerAbstract:Abstract Fibrinogen-binding proteins were found in the culture supernatants of Mannheimia haemolytica serotype 1 (ATCC 43270) and Pasteurella trehalosi serotype 10 (ECO-100). Sheep fibrinogen was biotinylated and shown to bind to proteins in the culture supernatants by modified western blot. Fibrinogen-binding proteins in the culture supernatant may be important virulence factors leading to the characteristic fibrinous pneumonia caused by these organisms and may be critical antigenic targets for immune prophylaxis.