Chronic exposure to coal fly ash causes minimal changes in corticosterone and testosterone concentrations in male southern toads Bufo terrestris.

More than 50% of the electricity in the United States is produced by coal-burning power plants. The byproduct of coal-burning plants is coal fly ash, which contains increased concentrations of trace metals and is disposed of in collection basins. Southern toads (Bufo terrestris) frequently use these basins for reproduction. Male toads were collected in spring 2001 and 2002 from an ash basin and a reference site and divided into four groups: toads collected at the control site and maintained on (1) control substrate and food or (2) ash and contaminated food and toads collected at the ash site and maintained in (3) control or (4) ash conditions. Blood was collected periodically during 5 months to determine testosterone and corticosterone concentrations. Reference to ash toads exhibited a significant, transient increase in corticosterone at 4 weeks, but neither corticosterone nor testosterone continued to increase beyond this time. In contrast, toads caught and maintained on ash did not exhibit increased corticosterone. Testosterone in these toads appeared to be unrelated to ash exposure. This unexpected lack of a corticosterone response and no effect on testosterone suggests that toads chronically exposed to trace metals can acclimate to a polluted environment, but they may still experience subtle long-term consequences.

Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC mutants in in vitro and in vivo systems.

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtC genes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously described H. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900-3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtA mutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.

Identification of the znuA-encoded periplasmic zinc transport protein of Haemophilus ducreyi.

The znuA gene of Haemophilus ducreyi encodes a 32-kDa (mature) protein that has homology to both the ZnuA protein of Escherichia coli and the Pzp1 protein of H. influenzae; both of these latter proteins are members of a growing family of prokaryotic zinc transporters. Inactivation of the H. ducreyi 35000 znuA gene by insertional mutagenesis resulted in a mutant that grew more slowly than the wild-type parent strain in vitro unless ZnCl(2) was provided at a final concentration of 100 microM. Other cations tested did not restore growth of this H. ducreyi mutant to wild-type levels. The H. ducreyi ZnuA protein was localized to the periplasm, where it is believed to function as the binding component of a zinc transport system. Complementation of the znuA mutation with the wild-type H. ducreyi znuA gene provided in trans restored the ability of this H. ducreyi mutant to grow normally in the absence of exogenously added ZnCl2. The wild-type H. ducreyi znuA gene was also able to complement a H. influenzae pzp1 mutation. The H. ducreyi znuA isogenic mutant exhibited significantly decreased virulence (P = 0.0001) when tested in the temperature-dependent rabbit model for experimental chancroid. This decreased virulence was not observed when the znuA mutant was complemented with the wild-type H. ducreyi znuA gene provided in trans.

Characterization of a Haemophilus ducreyi mutant deficient in expression of cytolethal distending toxin.

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that kills HeLa, HEp-2, and other human epithelial cells in vitro. H. ducreyi CDT activity is encoded by a three-gene cluster (cdtABC), and antibody to the cdtC gene product can neutralize CDT activity in vitro (L. D. Cope, S. R. Lumbley, J. L. Latimer, J. Klesney-Tait, M. K. Stevens, L. S. Johnson, M. Purven, R. S. Munson, Jr., T. Lagergard, J. D. Radolf, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 94:4056-4061, 1997). Culture supernatant fluid from a recombinant Escherichia coli strain containing the H. ducreyi cdtABC gene cluster readily killed both HeLa cells and HaCaT keratinocytes and had a modest inhibitory effect on the growth of human foreskin fibroblasts. Insertional inactivation of the cdtC gene in this recombinant E. coli strain eliminated the ability of this strain to kill HeLa cells and HaCaT keratinocytes. This mutated H. ducreyi cdtABC gene cluster was used to construct an isogenic H. ducreyi cdtC mutant. Monoclonal antibodies against the H. ducreyi CdtA, CdtB, and CdtC proteins were used to characterize protein expression by this cdtC mutant. Culture supernatant fluid from this H. ducreyi cdtC mutant did not detectably affect any of the human cells used in this study. The presence of the wild-type H. ducreyi cdtC gene in trans in this H. ducreyi mutant restored its ability to express a CDT that killed both HeLa cells and HaCaT keratinocytes. The isogenic H. ducreyi cdtC mutant was shown to be as virulent as its wild-type parent strain in the temperature-dependent rabbit model for experimental chancroid. Lack of expression of the H. ducreyi CdtC protein also did not affect the ability of this H. ducreyi mutant to survive in the skin of rabbits.

Effects of cyclic loading on selected post-and-core systems.

OBJECTIVE: The purpose of this study was to evaluate the resistance of selected post-and-core systems to fatigue testing. METHOD AND MATERIALS: A fatigue-testing machine and data acquisition software were developed for this in vitro test. Pulpless mandibular second premolars were treated with three different types of post-and-core system: (1) two different prefabricated posts with amalgam cores, (2) the same two prefabricated posts with resin composite cores, and (3) cast post and cores, which served as the control group. The fatigue-testing machine applied alternating buccal and lingual forces to the core until lateral movement of 0.0025 inches (63.5 microns) was detected by a transducer contacting the surface of the core. This criterion represented initial cement failure. RESULTS: Although groups using the resin composite core material exhibited a higher number of cycles prior to failure, there was no statistically significant difference between the different post-and-core systems. CONCLUSION: All of the post-and-core systems that were tested may be acceptable for restoring endodontically treated teeth.

Haemophilus ducreyi secretes a filamentous hemagglutinin-like protein.

We have identified two extremely large open reading frames (ORFs) in Haemophilus ducreyi 35000, lspA1 and lspA2, each of which encodes a predicted protein product whose N-terminal half is approximately 43% similar to the N-terminal half of Bordetella pertussis filamentous hemagglutinin (FhaB). To the best of our knowledge, lspA1 (12,500 nucleotides [nt]) and lspA2 (14,800 nt) are among the largest prokaryotic ORFs identified to date. The predicted proteins, LspA1 and LspA2, are 86% identical overall to each other and also have limited amino acid sequence similarity at their N termini to other secreted bacterial proteins, including certain hemolysins. Southern blot analysis indicated that lspA1 and lspA2 sequences were present in 15 other geographically diverse H. ducreyi strains. Reverse transcriptase PCR analysis of total RNA isolated from H. ducreyi 35000 grown in liquid medium, grown on solid agar medium, and isolated from lesions of H. ducreyi-infected rabbits indicated that lspA1 and lspA2 were transcribed both in vitro and in vivo. A 260-kDa protein present in culture supernatant from eight virulent H. ducreyi strains reacted with both polyclonal serum from rabbits infected with H. ducreyi 35000 and a monoclonal antibody predicted to bind both LspA1 and LspA2. This 260-kDa protein in H. ducreyi 35000 culture supernatant was shown to be the protein product of the lspA1 ORF based on its reactivity with a monoclonal antibody specific for LspA1. Four H. ducreyi strains, previously shown to be avirulent in the temperature-dependent rabbit model for chancroid, did not produce either LspA1 or LspA2 in vitro. This finding raised the possibility that LspA1, LspA2, or both may be involved in the ability of H. ducreyi to cause lesions in this animal model.

Detection and identification of Actinobacillus pleuropneumoniae serotype 5 by multiplex PCR.

Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 10(2) CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.

Cloning and mutagenesis of a serotype-specific DNA region involved in encapsulation and virulence of Actinobacillus pleuropneumoniae serotype 5a: concomitant expression of serotype 5a and 1 capsular polysaccharides in recombinant A. pleuropneumoniae serotype 1.

A DNA region involved in Actinobacillus pleuropneumoniae serotype 5 capsular polysaccharide (CP) biosynthesis was identified and characterized by using a probe specific for the cpxD gene involved in CP export. The adjacent serotype 5-specific CP biosynthesis region was cloned from a 5.8-kb BamHI fragment and an 8.0-kb EcoRI fragment of strain J45 genomic DNA. DNA sequence analysis demonstrated that this region contained four complete open reading frames, cps5A, cps5B, cps5C, and cps5D. Cps5A, Cps5B, and Cps5C showed low homology with several bacterial glycosyltransferases involved in the biosynthesis of lipopolysaccharide or CP. However, Cps5D had high homology with KdsA proteins (3-deoxy-D-manno-2-octulosonic acid 8-phosphate synthetase) from other gram-negative bacteria. The G+C content of cps5ABC was substantially lower (28%) than that of cps5D and the rest of the A. pleuropneumoniae chromosome (42%). A 2.1-kb deletion spanning the cloned cps5ABC open reading frames was constructed and transferred into the J45 chromosome by homologous recombination with a kanamycin resistance cassette to produce mutant J45-100. Multiplex PCR confirmed the deletion in this region of J45-100 DNA. J45-100 did not produce intracellular or extracellular CP, indicating that cps5A, cps5B, and/or cps5C were involved in CP biosynthesis. However, biosynthesis of the Apx toxins, lipopolysaccharide, and membrane proteins was unaffected by the mutation. Besides lack of CP biosynthesis, and in contrast to J45, J45-100 grew faster, was sensitive to killing in precolostral calf serum, and was avirulent in pigs at an intratracheal challenge dose three times the 50% lethal dose (LD50) of strain J45. At six times the J45 LD50, J45-100 caused mild to moderate lung lesions but not death. Electroporation of cps5ABC into A. pleuropneumoniae serotype 1 strain 4074 generated strain 4074(pJMLCPS5), which expressed both serotype 1 and serotype 5 CP. However, serotype 1 capsule expression was diminished in 4074(pJMLCPS5) in comparison to 4074. The recombinant strain produced significantly less total CP (serotypes 1 and 5 CP combined) in log phase (P = 0.0012) but significantly more total CP in late stationary phase than 4074 (P < 0.0001). In addition, strain 4074(pJMLCPS5) caused less mortality and bacteremia in pigs and mice following respiratory challenge than strain 4074, indicating that virulence was affected by diminished capsule production. These results emphasize the importance of CP in the serum resistance and virulence of A. pleuropneumoniae.

Identification and characterization of a DNA region involved in the export of capsular polysaccharide by Actinobacillus pleuropneumoniae serotype 5a.

Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide that acts as a protective barrier to phagocytosis and complement-mediated killing. To begin understanding the role of A. pleuropneumoniae capsule in virulence, we sought to identify the genes involved in capsular polysaccharide export and biosynthesis. A 5.3-kb XbaI fragment of A. pleuropneumoniae serotype 5a J45 genomic DNA that hybridized with DNA probes specific for the Haemophilus influenzae type b cap export region was cloned and sequenced. This A. pleuropneumoniae DNA fragment encoded four open reading frames, designated cpxDCBA. The nucleotide and predicted amino acid sequences of cpxDCBA contained a high degree of homology to the capsule export genes of H. influenzae type b bexDCBA, Neisseria meningitidis group B ctrABCD, and, to a lesser extent, Escherichia coli K1 and K5 kpsE and kpsMT. When present in trans, the cpxDCBA gene cluster complemented kpsM::TnphoA or kpsT::TnphoA mutations, determined by enzyme immunoassay and by restored sensitivity to a K5-specific bacteriophage. A cpxCB probe hybridized to genomic DNA from all A. pleuropneumoniae serotypes tested, indicating that this DNA was conserved among serotypes. These data suggest that A. pleuropneumoniae produces a group II family capsule similar to those of related mucosal pathogens.

Resistance of Actinobacillus pleuropneumoniae to bactericidal antibody and complement is mediated by capsular polysaccharide and blocking antibody specific for lipopolysaccharide.

Actinobacillus pleuropneumoniae is resistant to complement-mediated killing, even in the presence of specific Ab. Our studies focused on identifying the mechanism(s) responsible for this resistance. Encapsulated A. pleuropneumoniae was susceptible to killing in precolostral calf serum (PCS) but not in normal serum as a complement source in the presence of anti-capsular polysaccharide (CP) IgG. In contrast, two capsule-deficient mutants were sensitive to killing in normal serum and one was sensitive to killing in PCS alone. Electron microscopy demonstrated that A. pleuropneumoniae serotype 5a synthesized a thick, adherent CP that bound anti-CP Ab distant from the outer membrane. The CP of A. pleuropneumoniae did not prevent complement activation or the attachment of C3 to the cell surface. However, the CP did limit the amount of C9, a component of the membrane attack complex, that bound to A. pleuropneumoniae in PCS. A second mechanism of serum resistance was a result of an LPS-specific Ab present in the IgG fractions of normal swine serum, swine anti-K17 serum, and guinea pig anti-K17 LPS that blocked anti-CP IgG complement-mediated killing of A. pleuropneumoniae. Incubation of swine anti-K17 IgG with purified K17 LPS depleted Abs specific for K17 LPS but not for K17 proteins and removed all blocking activity. Immune swine serum containing this blocking Ab reduced the deposition of C9 on A. pleuropneumoniae in the presence of anti-CP IgG and also directed the deposition of C9 to sites on the bacteria in which the bound C9 was easily eluted. Thus, CP and anti-LPS Ab may act synergistically or at different stages of infection to limit the ability of complement to eliminate A. pleuropneumoniae.