Development of an algorithm for production of inactivated arbovirus antigens in cell culture.

Arboviruses are medically important pathogens that cause human disease ranging from a mild fever to encephalitis. Laboratory diagnosis is essential to differentiate arbovirus infections from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the virus-specific immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Antigen production in cell culture has largely replaced the use of suckling mice; however, the methods are not directly transferable. The development of a cell culture antigen production algorithm for nine arboviruses from the three main arbovirus families, Flaviviridae, Togaviridae, and Bunyaviridae, is described here. Virus cell culture growth and harvest conditions were optimized, inactivation methods were evaluated, and concentration procedures were compared for each virus. Antigen performance was evaluated by the MAC-ELISA at each step of the procedure. The antigen production algorithm is a framework for standardization of methodology and quality control; however, a single antigen production protocol was not applicable to all arboviruses and needed to be optimized for each virus.

Evidence of exposure to spotted fever group rickettsiae among Arizona dogs outside a previously documented outbreak area.

Since 2003, two communities in eastern Arizona have experienced a sustained outbreak of Rocky Mountain spotted fever (RMSF), caused by Rickettsia rickettsii, associated with transmission by Rhipicephalus sanguineus, the brown dog tick; 70 human cases, including eight deaths, were reported from these communities during 2003 through 2008. In both of the affected communities, antibodies to spotted fever group rickettsiae (SFGR) were present in dogs before the notice of the first human cases, suggesting that dogs may serve as useful sentinels for human risk of RMSF in this region. During 2005 and 2006, an exploratory serosurvey was conducted among stray and relinquished dogs presenting to animal control facilities in eastern Arizona located outside the area where human cases had been reported. Antibodies to SFGR were detected in 5.7% (14 of 247) dogs assessed outside the RMSF outbreak area. Animal shelters located in counties that either included or shared large borders with the outbreak area were significantly more likely to have seropositive dogs than facilities in more geographically separated counties (P = 0.01). In addition, stray dogs were significantly more likely to be antibody-positive than relinquished animals (P = 0.01), suggesting that control of stray dog populations should be considered as a means of limiting SFGR transmission in this region. The findings from this study may be extrapolated to suggest that the current risk for human RMSF infection may extend beyond the noted outbreak area. Heightened surveillance for human disease is needed in the region.

Incidence of respiratory pathogens in persons hospitalized with pneumonia in two provinces in Thailand.

Although pneumonia is a leading cause of death from infectious disease worldwide, comprehensive information about its causes and incidence in low- and middle-income countries is lacking. Active surveillance of hospitalized patients with pneumonia is ongoing in Thailand. Consenting patients are tested for seven bacterial and 14 viral respiratory pathogens by PCR and viral culture on nasopharyngeal swab specimens, serology on acute/convalescent sera, sputum smears and antigen detection tests on urine. Between September 2003 and December 2005, there were 1730 episodes of radiographically confirmed pneumonia (34·6% in children aged <5 years); 66 patients (3·8%) died. A recognized pathogen was identified in 42·5% of episodes. Respiratory syncytial virus (RSV) infection was associated with 16·7% of all pneumonias, 41·2% in children. The viral pathogen with the highest incidence in children aged <5 years was RSV (417·1/100,000 per year) and in persons aged ≥50 years, influenza virus A (38·8/100,000 per year). These data can help guide health policy towards effective prevention strategies.

Bartonella sp. bacteremia in patients with neurological and neurocognitive dysfunction.

We detected infection with a Bartonella species (B. henselae or B. vinsonii subsp. berkhoffii) in blood samples from six immunocompetent patients who presented with a chronic neurological or neurocognitive syndrome including seizures, ataxia, memory loss, and/or tremors. Each of these patients had substantial animal contact or recent arthropod exposure as a potential risk factor for Bartonella infection. Additional studies should be performed to clarify the potential role of Bartonella spp. as a cause of chronic neurological and neurocognitive dysfunction.

An outbreak of Rocky Mountain Spotted Fever associated with a novel tick vector, Rhipicephalus sanguineus, in Arizona, 2004: preliminary report.

This study describes preliminary results of an investigation of RMSF in Arizona associated with the brown dog tick, Rhipicephalus sanguineus. High numbers of dogs and heavy infestations of ticks created a situation leading to human disease.

A method for extracting RNA from dormant and germinating Bacillus subtilis strain 168 endospores.

RNA was extracted from dormant and germinating Bacillus subtilis 168 spores (intact spores and chemically decoated spores) by using rapid rupture followed by acid-phenol extraction. Spore germination progress was monitored by assaying colony forming ability before and after heat shock and by reading the optical density at 600 nm. The purity, yield, and composition of the extracted RNA were determined spectrophotometrically from the ratio of absorption at 260 nm to that at 280 nm; in a 2100 BioAnalyzer, giving the RNA yield/10(8) spores or cells and the distribution pattern of rRNA components. The method reported here for the extraction of RNA from dormant spores, as well as during different phases of germination and outgrowth, has proven to be fast, efficient and simple to handle. RNA of a high purity was obtained from dormant spores and during all phases of germination and growth. There was a significant increase in RNA yield during the transition from dormant spores to germination and subsequent outgrowth. Chemically decoated spores were retarded in germination and outgrowth compared with intact spores, and less RNA was extracted; however, the differences were not significant. This method for RNA isolation of dormant, germinating, and outgrowing bacterial endospores is a valuable prerequisite for gene expression studies, especially in studies on the responses of spores to hostile environmental conditions.

Roles of Bacillus endospores in the environment.

The occurrence and diverse roles of Bacillus spp. and their endospores in the environment is reviewed, with particular emphasis on soil ecology, host-symbiont and host-parasite interactions, and human exploitation of spores as biological control agents and probiotics.

Persistent infection in Neotoma fuscipes (Muridae: Sigmodontinae) with Ehrlichia phagocytophila sensu lato.

Dusky-footed wood rats (Neotoma fuscipes Baird) and two species of Peromyscus mice (P. maniculatus Wagner and P. truei Shufeldt) were collected over a 16-month period from three sites in Sonoma County, California. Blood was collected from 93 wood rats and 177 mice and serum or plasma was tested for seroreactivity with Ehrlichia phagocytophila sensu lato (also known as the human granulocytic ehrlichiosis agent). Thirty-five (37.6%) wood rats and 15 (8.5%) mice were seropositive. Positive Neotoma serology by site ranged from 9.4% to 62.1%. Polymerase chain reaction (PCR) testing for the Ehrlichia groESL heat shock operon was performed on all the seropositive and selected seronegative wood rats; 24 (68.6%) seropositive animals were PCR positive. Two seroconversions and no seroreversions were detected among 18 of the seropositive wood rats that were recaptured and tested multiple times (range = 2-6). Fourteen (77.8%) of the 18 were also PCR positive with six of these positive at every testing point (range = 2-6). One wood rat remained serologically and PCR positive in six specimens collected over a 14-month period. One male of 84 questing adult Ixodes pacificus Cooley & Kohls collected was PCR-positive for E. phagocytophila. Borrelia burgdorferi, the agent of Lyme disease, was cultured from ear punch biopsies from six of seven E. phagocytophila seropositive and one of four seronegative wood rats.

Clinical and serological follow-up of patients with human granulocytic ehrlichiosis in Slovenia.

An evaluation of the clinical outcome and the duration of the antibody response of patients with human granulocytic ehrlichiosis (HGE) was undertaken in Slovenia. Adult patients with a febrile illness occurring within 6 weeks of a tick bite were classified as having probable or confirmed HGE based on the outcome of serological or PCR testing. Thirty patients (median age, 44 years) were enrolled, and clinical evaluations and serum collection were undertaken at initial presentation and at 14 days, 6 to 8 weeks, and 3 to 4, 6, 12, 18, and 24 months. An indirect immunofluorescence assay (IFA) was performed, and reciprocal titers of > or =128 were interpreted as positive. Patients presented a median of 4 days after the onset of fever and were febrile for a median of 7.5 days; four (13.3%) received doxycycline. Seroconversion was observed in 3 of 30 (10.0%) patients, and 25 (83.3%) showed >4-fold change in antibody titer. PCR results were positive in 2 of 3 (66.7%) seronegative patients but in none of 27 seropositive patients at the first presentation. IFA antibody titers of > or =128 were found in 14 of 29 (48.3%), 17 of 30 (56.7%), 13 of 30 (43.4%), and 12 of 30 (40.0%) patients 6, 12, 18, and 24 months after presentation, respectively. Patients reporting additional tick bites during the study had significantly higher antibody titers at most time points during follow-up. No long-term clinical consequences were found during follow-up.

Prospective assessment of the etiology of acute febrile illness after a tick bite in Slovenia.

A prospective study established the etiology of febrile illnesses in residents of Slovenia that occurred within 6 weeks after a tick bite. A combination of laboratory and clinical criteria identified 64 (49.2%) of 130 patients as having confirmed, probable, or possible cases of tickborne disease during 1995 and 1996. Of the 130 patients, 36 (27.7%) had laboratory evidence of tickborne encephalitis, all of whom had clinically confirmed disease. Evidence of infection with Borrelia burgdorferi sensu lato was identified in 26 patients; 10 (7.7%) had confirmed Lyme borreliosis. Of 22 patients with evidence of Ehrlichia phagocytophila infection, 4 (3.1%) had confirmed ehrlichiosis. Infection by multiple organisms was found in 19 (14.6%) of 130 patients. Patients with meningeal involvement (43 [72.3%] of 59) were more likely to have confirmed tickborne disease than were patients with illness of undefined localization (18 [26.5%] of 68; P<.0001). Tickborne viral and bacterial infections are an important cause of febrile illness in Slovenia.

The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase.

The major DNA photoproduct of dormant, UV-irradiated Bacillus subtilis spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)]. During spore germination, SP is reversed to two intact thymines in situ by the DNA repair enzyme SP lyase, an S-adenosylmethionine (S-AdoMet)-dependent iron-sulfur ([Fe-S]) protein encoded by the splB gene. In the present work, cross-linking, SDS/PAGE, and size exclusion chromatography revealed that SplB protein dimerized when incubated with iron and sulfide under anaerobic reducing conditions. SplB isolated under aerobic conditions generated an EPR spectrum consistent with that of a partially degraded [3Fe-4S] center, and reduction of SplB with dithionite shifted the spectrum to that of a [4Fe-4S] center. Addition of S-AdoMet to SplB converted some of the [4Fe-4S] centers to an EPR-silent form consistent with electron donation to S-AdoMet. HPLC and electrospray ionization MS analyses showed that SP lyase cleaved S-AdoMet to generate 5'-deoxyadenosine. The results indicate that (i) SP lyase is a homodimer of SplB; (ii) dimer formation is coordinated by a [4Fe-4S] center; and (iii) the reduced [4Fe-4S] center is capable of donating electrons to S-AdoMet to generate a 5'-adenosyl radical that is then used for the in situ reversal of SP. Thus, SP lyase belongs to the "radical SAM" superfamily of enzymes that use [Fe-S] centers and S-AdoMet to generate adenosyl radicals to effect catalysis. SP lyase is unique in being the first and only DNA repair enzyme known to function via this novel enzymatic mechanism.

Role of dipicolinic acid in survival of Bacillus subtilis spores exposed to artificial and solar UV radiation.

Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) constitutes approximately 10% of Bacillus subtilis spore dry weight and has been shown to play a significant role in the survival of B. subtilis spores exposed to wet heat and to 254-nm UV radiation in the laboratory. However, to date, no work has addressed the importance of DPA in the survival of spores exposed to environmentally relevant solar UV radiation. Air-dried films of spores containing DPA or lacking DPA due to a null mutation in the DPA synthetase operon dpaAB were assayed for their resistance to UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight (290 to 400 nm), and sunlight from which the UV-B portion was filtered (325 to 400 nm). In all cases, air-dried DPA-less spores were significantly more UV sensitive than their isogenic DPA-containing counterparts. However, the degree of difference in UV resistance between the two strains was wavelength dependent, being greatest in response to radiation in the UV-B portion of the spectrum. In addition, the inactivation responses of DPA-containing and DPA-less spores also depended strongly upon whether spores were exposed to UV as air-dried films or in aqueous suspension. Spores lacking the gerA, gerB, and gerK nutrient germination pathways, and which therefore rely on chemical triggering of germination by the calcium chelate of DPA (Ca-DPA), were also more UV sensitive than wild-type spores to all wavelengths tested, suggesting that the Ca-DPA-mediated spore germination pathway may consist of a UV-sensitive component or components.

Fort Chaffee revisited: the epidemiology of tick-borne rickettsial and ehrlichial diseases at a natural focus.

A retrospective cohort study was conducted among troops training at Fort Chaffee, Arkansas, from May through June 1997, to identify infections caused by tick-borne pathogens. Serum samples were tested by IFAs for antibodies to selected Rickettsia and Ehrlichia species and by an investigational EIA for spotted fever group Rickettsia lipopolysaccharide antigens. Of 1,067 guardsmen tested, 162 (15.2%) had antibodies to one or more pathogens. Of 93 guardsmen with paired serum samples, 33 seroconverted to Rickettsia rickettsii or spotted fever group rickettsiae (SFGR) and five to Ehrlichia species. Most (84.8%) of the personnel who seroconverted to SFGR were detected only by EIA, and seropositivity was significantly associated with an illness compatible with a tick-borne disease. In addition, 34 (27%) of 126 subjects with detectable antibody titers reported a compatible illness. The primary risk factor for confirmed or probable disease was finding > 10 ticks on the body. Doxycycline use and rolling up of long sleeves were protective against seropositivity. The risk of transmission of tick-borne pathogens at Fort Chaffee remains high, and use of the broadly reactive EIA suggests that previous investigations may have underestimated the risk for infection by SFGR. Measures to prevent tick bite and associated disease may require reevaluation.

Temporal secretion of a multicellulolytic system in Myxobacter sp. AL-1. Molecular cloning and heterologous expression of cel9 encoding a modular endocellulase clustered in an operon with cel48, an exocellobiohydrolase gene.

The Gram-negative soil micro-organism Myxobacter sp. AL-1 possesses at least five extracellular cellulases, the production of which is regulated by the growth cycle. We cloned the complete gene for one of these cellulases, termed cel9, which encoded a 67-kDa modular family 9 endoglycohydrolase, which was produced during the stationary phase of growth and was strongly enhanced by avicel. The predicted product of cel9 matches the structural architecture of family 9 cellulases such as Thermonospora fusca endo/exocellulase E4. Cel9 protein was synthesized in Escherichia coli from a multicopy plasmid and in Bacillus subtilis from the isopropyl thiogalactoside-inducible Pspac promoter and was purified from the culture medium. Thermal stability, optimum pH and temperature dependence of Cel9 were similar when expressed from either source, and were indistinguishable from related cellulases produced by thermophilic bacteria. Downstream from cel9 was found a partial ORF, designated cel48, the deduced product of which was highly similar to bacterial exocellobiohydrolases and processive endoglucanases belonging to family 48 of the glycosyl hydrolases. The cel9 and cel48 genes appear to be arranged as part of an operon.

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.

Spore photoproduct (SP) lyase from Bacillus subtilis specifically binds to and cleaves SP (5-thyminyl-5,6-dihydrothymine) but not cyclobutane pyrimidine dimers in UV-irradiated DNA.

The predominant photolesion in the DNA of UV-irradiated dormant bacterial spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). A major determinant of SP repair during spore germination is its direct reversal by the enzyme SP lyase, encoded by the splB gene in Bacillus subtilis. SplB protein containing an N-terminal tag of six histidine residues [(6His)SplB] was purified from dormant B. subtilis spores and shown to efficiently cleave SP but not cyclobutane cis,syn thymine-thymine dimers in vitro. In contrast, SplB protein containing an N-terminal 10-histidine tag [(10His)SplB] purified from an Escherichia coli overexpression system was incompetent to cleave SP unless the 10-His tag was first removed by proteolysis at an engineered factor Xa site. To assay the parameters of binding of SplB protein to UV-damaged DNA, a 35-bp double-stranded oligonucleotide was constructed which carried a single pair of adjacent thymines on one strand. Irradiation of the oligonucleotide in aqueous solution or at 10% relative humidity resulted in formation of cyclobutane pyrimidine dimers (Py lozengePy) or SP, respectively. (10His)SplB was assayed for oligonucleotide binding using a DNase I protection assay. In the presence of (10His)SplB, the SP-containing oligonucleotide was selectively protected from DNase I digestion (half-life, >60 min), while the Py lozengePy-containing oligonucleotide and the unirradiated oligonucleotide were rapidly digested by DNase I (half-lives, 6 and 9 min, respectively). DNase I footprinting of (10His)SplB bound to the artificial substrate was carried out utilizing the (32)P end-labeled 35-bp oligonucleotide containing SP. DNase I footprinting showed that SplB protected at least a 9-bp region surrounding SP from digestion with DNase I with the exception of two DNase I-hypersensitive sites within the protected region. (10His)SplB also caused significant enhancement of DNase I digestion of the SP-containing oligonucleotide for at least a full helical turn 3' to the protected region. The data suggest that binding of SP lyase to SP causes significant bending or distortion of the DNA helix in the vicinity of the lesion.

Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments.

Endospores of Bacillus spp., especially Bacillus subtilis, have served as experimental models for exploring the molecular mechanisms underlying the incredible longevity of spores and their resistance to environmental insults. In this review we summarize the molecular laboratory model of spore resistance mechanisms and attempt to use the model as a basis for exploration of the resistance of spores to environmental extremes both on Earth and during postulated interplanetary transfer through space as a result of natural impact processes.