Biomedical subjects

W L Nicholson

Publications and source records attributed to W L Nicholson.

At least 37 records · Page 2Linked to original sources

PubMed2000

Detection of antibodies reactive with Ehrlichia chaffeensis in the raccoon.

Antibodies reactive with Ehrlichia chaffeensis were detected in raccoon (Procyon lotor) serum samples by using an indirect immunofluorescence assay. Samples from 411 raccoons trapped in the southeastern United States from 1977 to 1999 were tested. Serologically reactive samples with reciprocal titers of > or =16 were detected from 83 raccoons (20%) from 13 of 16 counties in eight states, indicating that raccoons are commonly exposed to E. chaffeensis. Samples collected as early as 1977 were positive. A polymerase chain reaction assay specific for E. chaffeensis failed to detect the presence of ehrlichial DNA in serum samples from 20 representative seroreactive raccoons. Because of serologic cross-reactivity among antigens derived from different Ehrlichia spp., additional immunologic, molecular, or culture-based studies will be required to confirm E. chaffeensis infections of raccoons in the southeastern United States.

Animals↗

PubMed1999

Shared vector-borne zoonoses of the Old World and New World: home grown or translocated?

Humans inhabiting the Old World and New World share a wide variety of pathogens. Processes that result in the disjunct biogeographic distribution of pathogens with common vertebrate reservoirs or vectors are more difficult to unravel than those influencing the distribution of infections spread only through human-to-human transmission. The origins of species and complexes of tick-borne bacteria are unclear. The agent of Lyme borreliosis may have speciated in the New World following geographical isolation of ticks harboring ancestral spirochetes; the subsequent spread to Europe of B. burgdorferi sensu stricto may have occurred within historical times. Other tick-borne agents, such as the ehrlichiae causing human granulocytic ehrlichiosis, are genetically very similar in the Old World and New World. As the taxonomic distinctions among these related agents of human and veterinary importance appear increasingly blurred, the processes leading to the current discontinuous geographic distributions will also become the source of continuing speculation. Accumulating data suggest an Old World origin for a group of bacteria that include B. elizabethae, a human pathogen first identified from the New World. The potential public health significance of these newly described organisms is undefined, but of international interest as their vertebrate reservoir has been introduced throughout the world.

Animals↗

PubMed1999

Method for purification of bacterial endospores from soils: UV resistance of natural Sonoran desert soil populations of Bacillus spp. with reference to B. subtilis strain 168.

Endospores of Bacillus spp. were purified from three Sonoran desert soil samples by Chelex extraction and NaBr density gradient centrifugation and their UV resistances compared with that of B. subtilis strain 168. Natural spore populations exhibited tight adherence to soil particles which was not readily overcome by the extraction and purification procedure. It was observed that spores purified from soil exhibited 2-3 fold higher resistance to UV (as measured by the 90% lethal dose, LD90) than did B. subtilis strain 168 grown on NSM, a standard laboratory sporulation medium, and purified by the same extraction procedure. Cultivation of spore-forming bacteria isolated from soil on NSM resulted in production of spores with essentially identical UV resistance as strain 168, suggesting that spore UV resistance is influenced by the environment in which spores are produced.

Bacillus↗

PubMed1999

Identity of ehrlichial DNA sequences derived from Ixodes ricinus ticks with those obtained from patients with human granulocytic ehrlichiosis in Slovenia.

Adult Ixodes ricinus (Acari: Ixodidae) ticks collected near Ljubljana, Slovenia, were tested for the agent of human granulocytic ehrlichiosis (HGE) by using PCR assays based on the 16S rRNA gene. Three (3.2%) of 93 ticks were found to contain granulocytic ehrlichiae. Nucleotide sequences of portions of the bacterial groESL heat shock operon amplified from these ticks were identical or nearly (99.8%) identical to those previously determined for human patients with HGE from Slovenia, providing additional evidence that the ticks were infected with the HGE agent. This study identified I. ricinus as the likely vector for these ehrlichial pathogens of humans in this part of Europe.

Animals↗

PubMed1999

Diagnosis of human ehrlichiosis by PCR assay of acute-phase serum.

A PCR assay of 43 acute-phase serum samples was evaluated as a method for early detection of human granulocytic ehrlichiosis (HGE) and determination of etiology when serologic testing is inconclusive. Sequence-confirmed products of the HGE agent were amplified from three individuals residing or having exposure history in Minnesota or Wisconsin, and similarly confirmed products from Ehrlichia chaffeensis were amplified from three individuals from Florida or Maryland. Etiology, as determined by PCR and serology, was the same whenever there was a fourfold difference between the maximum titers of antibodies to both antigens, indicating that presumptive determination of etiology may be based on fourfold differences in titers. PCR testing determined that E. chaffeensis was the etiologic agent for one individual who had similar titers of antibodies to both agents. PCR assay of acute-phase serum in the absence of whole blood specimens may be a useful method for early detection of human ehrlichiosis and determination of etiology when serologic testing is inconclusive.

Acute-Phase Reaction↗

PubMed1999

Dusky-footed wood rats (Neotoma fuscipes) as reservoirs of granulocytic Ehrlichiae (Rickettsiales: Ehrlichieae) in northern California.

Dusky-footed wood rats (Neotoma fuscipes) and Peromyscus sp. mice (P. maniculatus and P. truei) were collected from one site in Placer County, one site in Santa Cruz County, and two sites in Sonoma County in northern California. Serum or plasma samples from 260 rodents were tested for antibodies to the agent of human granulocytic ehrlichiosis. Of these, samples from 25 wood rats (34% of those tested) and 10 (8%) Peromyscus sp. mice were found to be seropositive, but only those from one site. PCR assays targeting the groESL heat shock operon were conducted on all seropositive specimens and a subset of seronegative blood specimens. Ehrlichial DNA was identified in 17 (68%) of the 25 seropositive wood rat blood samples and in 1 of the 10 (10%) Peromyscus sp. specimens. None of 40 seronegative blood samples was PCR positive. Both seropositive and PCR-positive animals were collected during each trapping period. One male tick out of 84 Ixodes pacificus adults collected was PCR positive; samples of Dermacentor occidentalis nymphs and adults were negative. Nucleotide sequences of amplicons from three wood rat blood specimens and from the single PCR-positive tick differed by one and two bases, respectively, from a sequence previously obtained from Ehrlichia equi. At one site in Sonoma County, wood rats had a concurrent high prevalence of seropositivity and PCR positivity, while other sigmodontine rodents collected at the site were only occasionally infected. We suggest that dusky-footed wood rats serve as reservoirs of granulocytic ehrlichial agents in certain areas of northern California. The tick species involved in the transmission of granulocytic ehrlichiae among wood rats remains unknown.

Animals↗

PubMed1999

Serologic testing for human granulocytic ehrlichiosis at a national referral center.

An indirect immunofluorescence assay (IFA) was used to identify patients with antibodies reactive to the human granulocytic ehrlichiosis (HGE) agent. Serum samples collected from clinically ill individuals were submitted to the Centers for Disease Control and Prevention by physicians via state health departments from throughout the United States and tested against a panel of ehrlichial and rickettsial pathogens. Antibodies reactive to the HGE agent were detected in 142 (8.9%) of 1,602 individuals tested. There were 19 confirmed and 59 probable (n = 78) cases of HGE as defined by seroconversion or a fourfold or higher titer to the HGE agent than to the Ehrlichia chaffeensis antigens. The average age of patients with HGE was 57 years, and males accounted for 53 (68%) of the patients. Cases of HGE occurred in 21 states; 47 (60%) of the cases occurred in Connecticut (n = 14), New York (n = 18), and Wisconsin (n = 15). Onset of HGE was identified from April through December, with cases peaking in June and July. The earliest confirmed cases of HGE occurred in 1987 in Wisconsin and 1988 in Florida. No fatalities were reported among the 78 patients with confirmed or probable HGE. Reactivity to the HGE agent and to either Coxiella burnetii, Rickettsia rickettsii, or Rickettsia typhi was infrequent; however, 74 (52%) of the 142 individuals who were positive for HGE had at least one serum sample that also reacted to the E. chaffeensis antigen. Thirty-four persons with confirmed or probable human monocytic ehrlichiosis due to E. chaffeensis also had antibodies to the HGE agent in at least one serum sample. The specific etiologic agent for 30 patients was not ascribed because of similarity of titers to both ehrlichial antigens. The use of both antigens may be required to correctly diagnose most cases of human ehrlichiosis, especially in geographic regions where both the HGE agent and E. chaffeensis occur.

Adolescent↗

PubMed1999

Outcome of diagnostic tests using samples from patients with culture-proven human monocytic ehrlichiosis: implications for surveillance.

We describe the concordance among results from various laboratory tests using samples derived from nine culture-proven cases of human monocytic ehrlichiosis (HME) caused by Ehrlichia chaffeensis. A class-specific indirect immunofluorescence assay for immunoglobulin M (IgM) and IgG, using E. chaffeensis antigen, identified 44 and 33% of the isolation-confirmed HME patients on the basis of samples obtained at initial clinical presentation, respectively; detection of morulae in blood smears was similarly insensitive (22% positive). PCR amplifications of ehrlichial DNA targeting the 16S rRNA gene, the variable-length PCR target gene, and the groESL operon were positive for whole blood specimens obtained from all patients at initial presentation. As most case definitions of HME require a serologic response with compatible illness for a categorization of even probable disease, PCR would have been required to confirm the diagnosis of HME in all nine of these patients without the submission of a convalescent-phase serum sample. These data suggest that many, if not most, cases of HME in patients who present early in the course of the disease may be missed and underscore the limitations of serologically based surveillance systems.

Antibodies, Bacterial↗

PubMed1998

Human ehrlichiosis in central Europe.

Ehrlichioses are tick-transmitted diseases associated with illnesses of animals for decades, but recently recognised to be emerging human diseases. In the last ten years increasing number of cases of human infections caused by Ehrlichia chaffeensis and granulo-cytic ehrlichia were described in the United States. Several reports also indicate the presence of infection with the human granulocytic ehrlichiosis (HGE) agent in Europe. The first confirmed acute human disease caused by HGE agent was reported from Slovenia. Until 1997, five patients have been discovered in a prospective study on the etiology of febrile illnesses occurring within six weeks following a tick bite, conducted at the Department of Infectious Diseases, University Medical Centre, Ljubljana, Slovenia. The diagnosis of acute HGE was established by seroconversion to the HGE agent and/or molecular identification of ehrlichial organisms. None of the patients had detectable morulae on blood smear examination. Clinical characteristics and laboratory findings were similar to those reported from the United States, although the disease course was relatively mild in the Slovenian cases. All patients recovered rapidly and without sequelae, although only three patients received antibiotic therapy (of whom only two were treated with doxycycline). Many ehrlichiosis cases could go undetected due to a lack of physician awareness, lack of public knowledge, or limited investigation. HGE should now be also included in the differential diagnosis of febrile illnesses occurring after a tick bite in Europe.

Acute Disease↗

PubMed1998

Human granulocytic ehrlichiosis in Europe: clinical and laboratory findings for four patients from Slovenia.

Febrile illnesses following a tick bite in patients from Slovenia were evaluated for an ehrlichial etiology. A case of acute human granulocytic ehrlichiosis (HGE) was confirmed by seroconversion to the HGE agent or molecular identification of ehrlichial organisms. Acute infection with the HGE agent was confirmed in four patients. None of the patients had detectable antibodies to the HGE agent at their first visit, but polymerase chain reaction analysis was positive for three patients. All four patients subsequently seroconverted to the HGE agent as shown by high titers of antibody. Clinical features and laboratory findings were similar to those in reports from the United States, although the disease course was relatively mild in the Slovenian cases. All patients recovered rapidly and without sequelae, although only two received antibiotic therapy (of whom only one was treated with doxycycline). HGE is an emerging tick-borne disease in the United States and should now be included in the differential diagnosis of febrile illnesses occurring after a tick bite in Europe.

Adult↗

PubMed1998

Spore photoproduct lyase from Bacillus subtilis spores is a novel iron-sulfur DNA repair enzyme which shares features with proteins such as class III anaerobic ribonucleotide reductases and pyruvate-formate lyases.

The major photoproduct in UV-irradiated spore DNA is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). An important determinant of the high UV resistance of Bacillus subtilis spores is the accurate in situ reversal of SP during spore germination by the DNA repair enzyme SP lyase. To study the molecular aspects of SP lyase-mediated SP repair, the cloned B. subtilis splB gene was engineered to encode SP lyase with a molecular tag of six histidine residues at its amino terminus. The engineered six-His-tagged SP lyase expressed from the amyE locus restored UV resistance to spores of a UV-sensitive mutant B. subtilis strain carrying a deletion-insertion mutation which removed the entire splAB operon at its natural locus and was shown to repair SP in vivo during spore germination. The engineered SP lyase was purified both from dormant B. subtilis spores and from an Escherichia coli overexpression system by nickel-nitrilotriacetic acid (NTA) agarose affinity chromatography and was shown by Western blotting, UV-visible spectroscopy, and iron and acid-labile sulfide analysis to be a 41-kDa iron-sulfur (Fe-S) protein, consistent with its amino acid sequence homology to the 4Fe-4S clusters in anaerobic ribonucleotide reductases and pyruvate-formate lyases. SP lyase was capable of reversing SP from purified SP-containing DNA in an in vitro reaction either when present in a cell-free extract prepared from dormant spores or after purification on nickel-NTA agarose. SP lyase activity was dependent upon reducing conditions and addition of S-adenosylmethionine as a cofactor.

Acetyltransferases↗

PubMed1998

Serologic evidence of infection with Ehrlichia spp. in wild rodents (Muridae: Sigmodontinae) in the United States.

Rodent (Muridae: Sigmodontinae) blood and sera collected from 14 states were tested for seroreactivity to a cultured isolate of the human granulocytic ehrlichiosis (HGE) agent by using an indirect immunofluorescence assay. Of the 1,240 samples tested, 136 (11%) were found to be reactive at titers of > or = 32. Rodents with HGE agent-specific antibodies were found in New York (23% of 491 samples; geometric mean endpoint titer [GMT] = 441), Connecticut (11% of 100 samples; GMT = 481), California (9% of 32 samples; GMT = 323), Colorado (2% of 212 samples; GMT = 256), Florida (7% of 27 samples; GMT = 362), Maryland (7% of 15 samples; titer = 64), New Jersey (4% of 76 samples; titer = 256), and Wisconsin (13% of 8 samples; titer = 128). Samples from Georgia (n = 16), Illinois (n = 27), Nevada (n = 27), North Carolina (n = 52), Ohio (n = 57), and Utah (n = 100) were not reactive. The earliest seroreactive sample was from a Peromyscus leucopus mouse collected in June 1986 in Connecticut, and the majority of the seroreactive samples (68%) were from this species. Samples from other Peromyscus species (P. boylii, P. maniculatus, and P. gossypinus) were also found to be reactive, with a GMT for the genus of 410. Several species of Neotoma woodrats (N. fuscipes, N. lepida, N. albigula, and N. mexicana) from California and Colorado had antibodies that reacted with the HGE agent (genus GMT = 194), suggesting that enzootic cycles of Ehrlichia spp. exist outside of the areas of confirmed human disease. Attempts to amplify and detect ehrlichial DNA from the limited tissues available (n = 40 animals) were unsuccessful. Further studies are needed to determine the identity of the organisms inducing antibody production in these rodent species and to elucidate the epidemiology and public health importance of these agents.

Animals↗

PubMed1998

Nested PCR assay for detection of granulocytic ehrlichiae.

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.

Animals↗

PubMed1997

Spore photoproduct lyase operon (splAB) regulation during Bacillus subtilis sporulation: modulation of splB-lacZ fusion expression by P1 promoter mutations and by an in-frame deletion of splA.

EsigmaG-dependent transcription of the splAB operon in the forespore at stage III of Bacillus subtilis sporulation initiates from two promoters, P1 preceding splA (major) and P3 preceding splB (minor). To explore the possible role of splA in controlling splB-encoded spore photoproduct lyase expression, we measured beta-galactosidase from splB-lacZ fusions integrated at the SPbeta prophage locus which contained point mutations or deletions which either inactivated or physically removed P1 and/or splA. Paradoxically, inactivation of P1 by point mutation or its removal by deletion from upstream resulted in elevated beta-galactosidase expression of the resulting splB-lacZ fusion, as did an in-frame deletion of splA which left P1 and P3 intact;however, expression of all fusions remained sporulation specific and EsigmaG dependent.

Bacillus subtilis↗

PubMed1997

Analysis of spore photoproduct lyase operon (splAB) function using targeted deletion-insertion mutations spanning the Bacillus subtilis operons ptsHI and splAB.

Germinating Bacillus subtilis spores repair UV-induced DNA damage in part using the enzyme spore photoproduct (SP) lyase. SP lyase is encoded by splB, the second cistron of the splAB operon. The splAB operon is transcribed during sporulation from the P1 promoter, which partially overlaps the transcriptional terminator of the upstream ptsHI operon, which in turn encodes the Hpr protein and Enzyme I components of the PEP:sugar phosphotransferase (PTS) system. In order to determine the physical and functional boundaries of these contiguous operons, null mutations were generated in the region by in vitro site-directed mutagenesis, in which parts of the cloned ptsI-splAB region were removed and replaced with an ermC antibiotic resistance cassette, then introduced by transformation into B. subtilis. A deletion-insertion spanning ptsI, splA, and splB abolished the ability of the resulting mutant to utilize the PTS sugar glucose. Deletions removing either splB alone or both splA and splB did not affect glucose utilization, thus indicating that splAB gene products are not involved in PTS function. A complementation system was developed using the deletion-insertion mutant lacking splAB which allows placement of alleles of the cloned splAB operon at the chromosomal amyE locus. The complementation system was used to explore the role of SP lyase in determining spore UV resistance.

Amino Acid Sequence↗

PubMed1997

An indirect immunofluorescence assay using a cell culture-derived antigen for detection of antibodies to the agent of human granulocytic ehrlichiosis.

An indirect immunofluorescence assay for the detection of human antibodies to the agent of human granulocytic ehrlichiosis (HGE) was developed and standardized. Antigen was prepared from a human promyelocytic leukemia cell line (HL-60) infected with a tick-derived isolate of the HGE agent (USG3). Suitable antigen presentation and preservation of cellular morphology were obtained when infected cells were applied and cultured on the slide, excess medium was removed, and cells were fixed with acetone. Use of a buffer containing bovine serum albumin and goat serum reduced background fluorescence, and use of an immunoglobulin G (gamma-specific) conjugate reduced nonspecific binding. The assay readily detected specific antibody from HGE patients and did not detect antibody from healthy individuals. No significant reactivity was noted in sera from patients with high titers of antibodies to other rickettsial species. We were able to identify antibodies reactive to USG3 antigen in samples from areas where HGE is endemic that had tested negative to other rickettsial agents. Animal sera reactive against Ehrlichia equi or Ehrlichia phagocytophila bound to the HGE antigen, indicating that the assay may be useful for veterinary use. Comparability between two different laboratories was assessed by using coded human sera exchanged between laboratories. Results from the two laboratories were similar, indicating that the assay can be easily integrated into use for routine testing for HGE. The assay was then compared to an assay using horse neutrophils infected with ehrlichiae. The two assays gave comparable results, indicating that the cell culture-derived antigen can be used for testing samples that have been previously tested with E. equi as an antigen. The new assay offers several advantages over other immunofluorescence methods that use animal-derived antigen and is suitable for use in testing for human antibodies to the HGE agent.

Animals↗

PubMed1997

PCR amplification and comparison of nucleotide sequences from the groESL heat shock operon of Ehrlichia species.

Degenerate PCR primers derived from conserved regions of the eubacterial groESL heat shock operon were used to amplify groESL sequences of Ehrlichia equi, Ehrlichia phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), Ehrlichia canis, Bartonella henselae, and Rickettsia rickettsii. The groESL nucleotide sequences were less conserved than the previously determined 16S rRNA gene sequences of these bacteria. A phylogenetic tree derived from deduced GroEL amino acid sequences was similar to trees based on 16S rRNA gene sequences. Nucleotide sequences obtained from clinical samples containing E. equi, E. phagocytophila, or the HGE agent were very similar (99.9 to 99.0% identity), and the deduced amino acid sequences were identical. Some divergence was evident between nucleotide sequences amplified from samples originating from the United States (E. equi and the HGE agent) and sequences from the European species, E. phagocytophila. A single pair of PCR primers derived from these sequences was used to detect E. chaffeensis and HGE agent DNA in blood samples from human patients with ehrlichiosis.

Base Sequence↗

PubMed1996

The two major spore DNA repair pathways, nucleotide excision repair and spore photoproduct lyase, are sufficient for the resistance of Bacillus subtilis spores to artificial UV-C and UV-B but not to solar radiation.

Bacterial endospores are 1 to 2 orders of magnitude more resistant to 254-nm UV (UV-C) radiation than are exponentially growing cells of the same strain. This high UV resistance is due to two related phenomena: (i) DNA of dormant spores irradiated with 254-nm UV accumulates mainly a unique thymine dimer called the spore photoproduct (SP), and (ii) SP is corrected during spore germination by two major DNA repair pathways, nucleotide excision repair (NER) and an SP-specific enzyme called SP lyase. To date, it has been assumed that these two factors also account for resistance of bacterial spores to solar UV in the environment, despite the fact that sunlight at the Earth's surface consists of UV-B, UV-A, visible, and infrared wavelengths of approximately 290 nm and longer. To test this assumption, isogenic strains of Bacillus subtilis lacking either the NER or SP lyase DNA repair pathway were assayed for their relative resistance to radiation at a number of UV wavelengths, including UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight, and sunlight from which the UV-B portion had been removed. For purposes of direct comparison, spore UV resistance levels were determined with respect to a calibrated biological dosimeter consisting of a mixture of wild-type spores and spores lacking both DNA repair systems. It was observed that the relative contributions of the two pathways to spore UV resistance change depending on the UV wavelengths used in a manner suggesting that spores irradiated with light at environmentally relevant UV wavelengths may accumulate significant amounts of one or more DNA photoproducts in addition to SP. Furthermore, it was noted that upon exposure to increasing wavelengths, wild-type spores decreased in their UV resistance from 33-fold (UV-C) to 12-fold (UV-B plus UV-A sunlight) to 6-fold (UV-A sunlight alone) more resistant than mutants lacking both DNA repair systems, suggesting that at increasing solar UV wavelengths, spores are inactivated either by DNA damage not reparable by the NER or SP lyase system, damage caused to photosensitive molecules other than DNA, or both.

Bacillus subtilis↗