Strategic selection of an optimal sorbent mixture for in-situ remediation of heavy metal contaminated sediments: framework and case study.

Aquatic sediments contaminated with heavy metals originating from mining and metallurgical activities pose significant risk to the environment and human health. These sediments not only act as a sink for heavy metals, but can also constitute a secondary source of heavy metal contamination. A variety of sorbent materials has demonstrated the potential to immobilize heavy metals. However, the complexity of multi-element contamination makes choosing the appropriate sorbent mixture and application dosage highly challenging. In this paper, a strategic framework is designed to systematically address the development of an in-situ sediment remediation solution through Assessment, Feasibility and Performance studies. The decision making tools and the experimental procedures needed to identify optimum sorbent mixtures are detailed. Particular emphasis is given to the utilization and combination of commercially available and waste-derived sorbents to enhance the sustainability of the solution. A specific case study for a contaminated sediment site in Northern Belgium with high levels of As, Cd, Pb and Zn originating from historical non-ferrous smelting is presented. The proposed framework is utilized to achieve the required remediation targets and to meet the imposed regulations on material application in natural environments.

Direct inoculation of Mycobacterium avium subspecies Paratuberculosis into ileocecal Peyer's patches results in colonization of the intestine in a calf model.

The objective of this study was to develop an intestinal model of Mycobacterium avium subspecies paratuberculosis (Map) infection in the calf for evaluation of mucosal pathology and local and systemic immunologic responses. Map was inoculated into Peyer's patches of young calves using a right flank surgical approach in standing calves to exteriorize the ileocecal junction. Inoculum doses ranging from 10(3) to 10(9) colony-forming units of strain K10 Map were injected through the serosal surface into Peyer's patches of the distal ileum near the ileocecal valve. Fecal samples were collected for culture from each calf weekly until termination of the study. Calves were necropsied at 7, 30, 60, and 90 days after infection, when inoculation sites, lymph nodes, spleen, and peripheral blood were collected for evaluation. Ileocecal lymph nodes were consistently colonized by Map in the 10(5) to 10(9) groups. The ileocecal valve was also colonized in 10(7) and 10(9) groups. This correlated with fecal culture results as infected calves intermittently shed Map in their feces throughout the study. Granulomatous lesions with giant cells and acid-fast bacilli at the ileocecal junction, ileocecal lymph nodes, and lamina propria of high-dose animals (10(7) and 10(9)) were identified from each time point. Flow cytometry was used to detect antigen-specific production of interferon-γ and interleukin-4 locally (ileocecal lymph node) and systemically (peripheral blood mononuclear cells), which defined distinct immunologic profiles in low-dose and high-dose calves. This study demonstrates intestinal Map infection via Peyer's patch inoculation, a novel model with many shared features of natural Map infection.

An equine protozoal myeloencephalitis challenge model testing a second transport after inoculation with Sarcocystis neurona sporocysts.

Previous challenge studies performed at Ohio State University involved a transport-stress model where the study animals were dosed with Sarcocystis neurona sporocysts on the day of arrival. This study was to test a second transportation of horses after oral inoculation with S. neurona sporocysts. Horses were assigned randomly to groups: group 1, transported 4 days after inoculation (DAI); group 2, at 11 DAI; group 3, at 18 DAI; and group 4, horses were not transported a second time (controls). An overall neurologic score was determined on the basis of a standard numbering system used by veterinarians. All scores are out of 5, which is the most severely affected animal. The mean score for the group 1 horses was 2.42; group 2 horses was 2.5; group 3 horses was 2.75; and group 4 horses was 3.25. Because the group 4 horses did not have a second transport, they were compared with all other groups. Statistically different scores were present between group 4 and groups 1 and 2. There was no difference in the time of seroconversion between groups. There was a difference between the time of onset of first clinical signs between groups 1 and 4. This difference was likely because of the different examination days. Differences in housing and handling were likely the reason for the differences in severity of clinical signs. This model results in consistent, significant clinical signs in all horses at approximately the same time period after inoculation but was most severe in horses that did not experience a second transport.

Equine vaccine for West Nile virus.

To meet the urgent need of controlling West Nile virus (WNV) infection in the equine population, we have developed a killed WNV vaccine. A dose titration study in horses was first conducted to evaluate serum neutralization antibody responses against WNV in these animals. Horses were vaccinated intramuscularly twice with the test vaccine at low, medium and high dose, three weeks apart. Serum samples were collected periodically and were measured for serum neutralizing antibody using a plaque reduction neutralization test. Significant increases in serum neutralizing antibody were detected in all three dosage groups 14 days post the second vaccination. Twelve months after the second vaccination, horses vaccinated with the medium dose of WNV vaccine and non-vaccinated control horses were experimentally challenged with WNV. Nine out of 11 (81.8%) controls developed viraemia after challenge while only one out of 19 (5.3%) vaccinates had transient viraemia, representing a 94% preventable fraction. In a separate study, the safety of the killed WNV vaccine was demonstrated under field conditions. A total of 648 horses, including 32 pregnant mares, were enrolled in the study. During the two weeks post vaccination period, no local or systemic adverse reactions were observed following 96% of the vaccinations administered while mild, transient injection site reactions were noted in a small number of horses. These results indicate that the killed WNV vaccine developed by Fort Dodge Animal Health is safe and efficacious.

Experimental induction of chronic borreliosis in adult dogs exposed to Borrelia burgdorferi-infected ticks and treated with dexamethasone.

OBJECTIVE: To develop a method to experimentally induce Borrelia burgdorferi infection in young adult dogs. ANIMALS: 22 healthy Beagles. PROCEDURE: All dogs were verified to be free of borreliosis. Twenty 6-month-old dogs were exposed to Borrelia burgdorferi-infected adult ticks and treated with dexamethasone for 5 consecutive days. Two dogs not exposed to ticks were treated with dexamethasone and served as negative-control dogs. Clinical signs, results of microbial culture and polymerase chain reaction (PCR) testing, immunologic responses, and gross and histologic lesions were evaluated 9 months after tick exposure. RESULTS: Predominant clinical signs were episodic pyrexia and lameness in 12 of 20 dogs. Infection with B burgdorferi was detected in microbial cultures of skin biopsy specimens and various tissues obtained during necropsy in 19 of 20 dogs and in all 20 dogs by use of a PCR assay. All 20 exposed dogs seroconverted and developed chronic nonsuppurative arthritis. Three dogs also developed mild focal meningitis, 1 dog developed mild focal encephalitis, and 18 dogs developed perineuritis or rare neuritis. Control dogs were seronegative, had negative results for microbial culture and PCR testing, and did not develop lesions. CONCLUSIONS AND CLINICAL RELEVANCE: Use of this technique successfully induced borreliosis in young dogs. Dogs with experimentally induced borreliosis may be useful in evaluating vaccines, chemotherapeutic agents, and the pathogenesis of borreliosis-induced arthritis.

Predicting the reactivity of proteins from their sequence alone: Kazal family of protein inhibitors of serine proteinases.

An additivity-based sequence to reactivity algorithm for the interaction of members of the Kazal family of protein inhibitors with six selected serine proteinases is described. Ten consensus variable contact positions in the inhibitor were identified, and the 19 possible variants at each of these positions were expressed. The free energies of interaction of these variants and the wild type were measured. For an additive system, this data set allows for the calculation of all possible sequences, subject to some restrictions. The algorithm was extensively tested. It is exceptionally fast so that all possible sequences can be predicted. The strongest, the most specific possible, and the least specific inhibitors were designed, and an evolutionary problem was solved.

Binding of amino acid side-chains to S1 cavities of serine proteinases.

The P1 or primary specificity residue of standard mechanism canonical protein inhibitors of serine proteinases, inserts into the S1 primary specificity cavity of the cognate enzyme upon enzyme-inhibitor complex formation. Both natural evolution and protein engineering often change the P1 residue to greatly alter the specificity and the binding strength. To systematize such results we have obtained all 20 coded P1 variants of one such inhibitor, turkey ovomucoid third domain, by recombinant DNA technology. The variants were extensively characterized. The association equilibrium constants were measured at pH 8.30, 21 (+/-2) degrees C, for interaction of these variants with six well characterized serine proteinases with hydrophobic S1, cavities. The enzyme names are followed by the best, worst and most specific coded residue for each. Bovine chymotrypsin A alpha (Tyr, Pro, Trp), porcine pancreatic elastase (Leu/Ala, Arg, Ala), subtilisin Carlsberg (Cys, Pro, Glu), Streptomyces griseus proteinase A (Cys, Pro, Leu) and B (Cys, Pro, Lys) and human leukocyte elastase (Ile, Asp, Ile). The data set was merged with Ka values for five non-coded variants at P1 of turkey ovomucoid third domain obtained in our laboratory by enzymatic semisynthesis. The ratios of the highest to the lowest Ka for each of the six enzymes range from 10(6) to 10(8). The dominant force for binding to these pockets is the hydrophobic interaction. Excess steric bulk (too large for the pocket), awkward shape (Pro, Val and Ile), polarity (Ser) oppose interaction. Ionic charges, especially negative charges on Glu- and Asp- are strongly unfavorable. The Pearson pro duct moment correlations for all the 15 enzyme pairs were calculated. We suggest that these may serve as a quantitative description of the specificity of the enzymes at P1. The sets of Streptomyces griseus proteinases A and B and of the two elastases are strongly positively correlated. Strikingly, chymotrypsin and pancreatic elastase are negatively correlated (-0.10). Such correlations can be usefully extended to many other enzymes and to many other binding pockets to provide a general measure of pocket binding specificity.

Activation of bovine neutrophils by recombinant bovine tumor necrosis factor-alpha.

The in vitro effect of bovine recombinant tumor necrosis factor-alpha (rbTNF-alpha) on bovine neutrophil function and the possibility that rbTNF-alpha and recombinant bovine interferon-gamma (rbIFN-gamma) act synergistically were investigated. Treatment of neutrophils with rbTNF-alpha (0.05 micrograms/ml; approximately 50 U/ml) at 37 degrees C for 2.5 h resulted in enhancement of antibody independent neutrophil-mediated cytotoxicity (AINC) and inhibition of random migration and chemotaxis. The same treatment resulted in a slight decrease in iodination and cytochrome C reduction, but did not affect Staphylococcus aureus ingestion, or antibody dependent cell-mediated cytotoxicity. Kinetic and inhibitor studies indicated that the action of rbTNF-alpha was rapid and was independent of protein and RNA synthesis by neutrophils. Evaluation of the synergistic activities of rbTNF-alpha and rbIFN-gamma indicated that treatment of neutrophils with these two cytokines simultaneously resulted in additive enhancement of AINC and inhibition of random migration and chemotaxis. There was no additive effect of the two cytokines on inhibition of iodination or cytochrome C reduction.

Improved protection of swine from pleuropneumonia by vaccination with proteinase K-treated outer membrane of Actinobacillus (Haemophilus) pleuropneumoniae.

The immunogenic and protective potentials of an outer membrane-enriched fraction (OM) from a serotype 5 strain of Actinobacillus (Haemophilus) pleuropneumoniae (APP) and the same OM degraded with proteinase K or periodate were evaluated in swine. Groups of pigs were vaccinated with two doses of OM, proteinase K-treated OM (P-OM), periodate-treated OM (PI-OM), or placebo vaccine and challenged intranasally with the homologous strain of APP. Results from triplicate experiments indicated that proteinase K treatment of OM resulted in an improved efficacy. This improved efficacy of P-OM vaccine over untreated OM vaccine was evidenced not only by less severe lung lesions in P-OM vaccinated pigs but also by significant reduction (P less than 0.05) in the number of P-OM vaccinated pigs which developed lung lesions upon challenge with APP. Assessment of sera from vaccinated animals by immunoblotting, complement fixation test, or ELISA indicated that the immunogenicity of some but not all protein or carbohydrate components were reduced (or eliminated) by proteinase K and periodate treatments respectively.

Influence of recombinant bovine interferon gamma and dexamethasone on pneumonia attributable to Haemophilus somnus in calves.

The influence of recombinant bovine interferon gamma (rBoIFN-gamma) treatment on resistance of clinically normal and dexamethasone-treated calves to Haemophilus somnus infection was evaluated. Four groups of 6 calves each were treated with saline solution (controls), dexamethasone (0.04 mg/kg of body weight/for 3 days), rBoIFN-gamma (2 micrograms/kg for 2 days), or dexamethasone and rBoIFN-gamma (aforementioned dosages). All treatments were started 24 hours before intrabronchial challenge exposure with 5 x 10(9) colony-forming units of H somnus. Rectal temperature and WBC count were monitored daily. Two of the dexamethasone-treated calves died of pneumonia 4 days after challenge exposure and were necropsied. All other calves were euthanatized and necropsied 7 days after challenge exposure. All calves had pneumonia of variable intensity. Dexamethasone-treated calves had increased volume of pneumonic lung (P less than 0.05) and increased severity of pneumonia, compared with control calves. Recombinant bovine interferon gamma treatment resulted in reduction in pneumonic lung volume and severity of pneumonia in dexamethasone-treated calves (P less than 0.05), although it did not influence severity of pneumonia in nondexamethasone-treated calves.

Identification of suppressive components in "Haemophilus somnus" fractions which inhibit bovine polymorphonuclear leukocyte function.

"Haemophilus somnus" fractions which inhibited iodination of protein by bovine polymorphonuclear leukocytes were isolated by heat extracting a washed bacterial suspension at 60 degrees C or incubating the bacterial suspension at 37 degrees C and were partially purified by ultrafiltration. The components in each fraction were separated by reverse-phase high-performance liquid chromatography and identified as ribonucleotides, a ribonucleoside, and purine and pyrimidine bases. Most of the compounds were found to be inhibitory to iodination in a dose-dependent manner. When the effect of each component on iodination at the concentrations present in "H. somnus" fractions was determined, it was found that guanine and GMP were the components responsible for most of the suppression in the fraction isolated by heat extraction, whereas guanine and adenine were the major inhibitory components in the fraction isolated by incubation at 37 degrees C.

Haemophilus somnus-induced interference with bovine neutrophil functions.

The effect of Haemophilus somnus on bovine polymorphonuclear leukocyte (PMN) function was examined in vitro with whole cells and fractions extracted from the surface of this bacterium. The ability of PMNs to iodinate protein and ingest Staphylococcus aureus was significantly inhibited in the presence of live cells, heat-killed whole cells or supernatant fluid from heat-killed cells, but not in the presence of washed, heat-killed cells. None of the fractions inhibited nitroblue tetrazolium (NBT) reduction by PMNs. The PMN inhibitory factors were further characterized. The material that inhibited S. aureus ingestion was found to be a heat-stable cell surface material of greater than 300 000 MW. The fraction inhibiting iodination of protein was found to be less than 10 000 MW.

Response of cultured IEC-17 normal rat intestinal epithelial cells to X radiation.

The growth parameters and radiosensitivity of normal rat intestinal epithelial cells, IEC-17, were studied. The cells were cultured by standard methods and exposed to an array of doses (1-12 Gy) of 250 kVp X rays. The survival curves generated exhibited no initial shoulder and were bimodal. The Do of the first component was about 0.2 Gy and the second component. 5.0 Gy. The ability of this cell line to repair sublethal lesions was examined by fractionation studies; repair was completed within 60 min after the first dose. When Chinese hamster ovary (CHO) cells were grown under the same conditions used for the IEC-17 cells and then irradiated with single doses, a typical survival curve with a Do of 1.4 Gy was obtained. The survival curves obtained for the IEC-17 cell line are consistent with the response of a morphologically distinct single population containing two functionally separate types of cells.

Restriction fragments that exert promoter activity during postexponential growth of Bacillus subtilis.

Two restriction fragments of Bacillus subtilis DNA were identified which caused the cat-86 gene present on the promoter cloning plasmid pPL703 to be activated predominantly during postexponential growth of host cells. The postexponential increase was observed in both sporulation-positive strains and in a spoOA mutant of B. subtilis. However, the postexponential increase in the cat-86 gene product, chloramphenicol acetyltransferase, was diminished or not observed when the plasmid-containing cells were grown in the presence of excess glucose. The promoter-containing fragment, designated as 33, was mapped to a site on the B. subtilis chromosome adjacent to hisA. The other fragment, 14, mapped to a site adjacent to ctrA. When present on a high-copy vector, both fragments caused a reduction in the sporulation frequency of host cells. Fragment 33 in high copy number conferred on B. subtilis cells three additional phenotypic changes: brown colony color, intracellular inclusions, and, in a protease-deficient mutant, the production of extracellular protease activity. These activities were observed only in postexponential-phase cultures.