Toxicity of isocycloseram, an isoxazoline insecticide, against laboratory and field-collected German cockroaches (Blattodea: Ectobiidae).

Isocycloseram is a new insecticide in the isoxazoline class that targets insect GABA-gated chloride channels. In this study, we evaluated a cockroach gel bait formulation containing 1% isocycloseram against a susceptible strain (UCR) and 5 field-collected strains (WM, RG386, Ryan, CDR, and SY) of the German cockroach, Blattella germanica (L.) (Blattodea: Ectobiidae), and compared it with several commercial insecticide baits in the laboratory. Using the Ebeling choice box method, we also tested a residual deposit of an SC formulation of isocycloseram against the UCR, RG386, and Ryan strains. The isocycloseram bait was among the fastest-performing treatments against adult males (mean survival time: 0.9-2.7 days) and mixed stages and sexes (mean survival time: 1.4-5.4 days) across all strains. Secondary transfer effects of the bait were demonstrated in the UCR strain by exposing new adult males to individuals killed by direct bait treatment. Physiological resistance was not detected in the WM, CDR, and RG386 strains with topical treatment of a diagnostic dose (3× LD95) of isocycloseram developed using the UCR strain. However, topical assays revealed resistance ratios (RR50) of 1.6 and 3.0× in the Ryan and SY strains, respectively. The performance of a 0.05% isocycloseram residual application against the Ryan strain was improved with the addition of piperonyl butoxide.

Mortality Rate of House Flies (Diptera: Muscidae) Exposed to Insecticidal Granular Fly Baits Containing Indoxacarb, Dinotefuran, or Cyantraniliprole.

The mortality rate of a field population of house fly (Musca domestica L.) was determined for a granular fly bait containing the active ingredient indoxacarb, which was compared to two commercially available granular fly baits containing either dinotefuran or cyantraniliprole. Indoxacarb was applied at three different application rates 0.498, 0.986, and 1.972 g/m2 (low, medium, and high). Time to 50% mortality was fastest for dinotefuran (5.7 h) and slowest for the low application rate of indoxacarb (10.3 h). Time to 90% mortality was fastest for the high application rate of indoxacarb (27.7 h) and slowest for dinotefuran (51.0 h) and cyantraniliprole (45.9 h). Among the three indoxacarb application rates, the high rate reached both 50 and 90% fly mortality significantly faster than the low rate. The medium rate did not significantly differ from either the high or low application rates. Dinotefuran bait produced greater fly mortality than all other treatments at 30-min post-exposure, with mortality for remaining baits exceeding controls by 3- to 6-h post-exposure. All insecticidal baits produced similar fly mortality by 6-h post-exposure and >94% fly mortality by 96-h post-exposure, indicating that each may be effective in a fly management program. Flies consumed a similar amount of the indoxacarb (regardless of application rate) and dinotefuran baits, but consumed less of the cyantraniliprole bait, suggesting a feeding irritancy or toxicity effect manifested during consumption. Nevertheless, flies consumed enough cyantraniliprole bait to cause mortality similar to other baits by 6-h post-exposure.

Evaluation of a digital flocculation reader for the rapid plasma reagin test for the serological diagnosis of syphilis.

We described the ASiManager-AT digital flocculation reader to demonstrate concordance between visual and digital readings of the rapid plasma reagin test for detection of antibodies in the serum of patients with syphilis. A qualitative and quantitative rapid plasma reagin was performed on each serum samples giving a concordance of 98.6% and 99.7%, respectively, for reactives and 100% for nonreactives.

Surface immunolabeling and consensus computational framework to identify candidate rare outer membrane proteins of Treponema pallidum.

Treponema pallidum reacts poorly with the antibodies present in rabbit and human syphilitic sera, a property attributed to the paucity of proteins in its outer membrane. To better understand the basis for the syphilis spirochete's "stealth pathogenicity," we used a dual-label, 3-step amplified assay in which treponemes encapsulated in gel microdroplets were probed with syphilitic sera in parallel with anti-FlaA antibodies. A small (approximately 5 to 10%) but reproducible fraction of intact treponemes bound IgG and/or IgM antibodies. Three lines of evidence supported the notion that the surface antigens were likely ß-barrel-forming outer membrane proteins (OMPs): (i) surface labeling with anti-lipoidal (VDRL) antibodies was not observed, (ii) immunoblot analysis confirmed prior results showing that T. pallidum glycolipids are not immunoreactive, and (iii) labeling of intact organisms was not appreciably affected by proteinase K (PK) treatment. With this method, we also demonstrate that TprK (TP0897), an extensively studied candidate OMP, and TP0136, a lipoprotein recently reported to be surface exposed, are both periplasmic. Consistent with the immunolabeling studies, TprK was also found to lack amphiphilicity, a characteristic property of ß-barrel-forming proteins. Using a consensus computational framework that combined subcellular localization and ß-barrel structural prediction tools, we generated ranked groups of candidate rare OMPs, the predicted T. pallidum outer membrane proteome (OMPeome), which we postulate includes the surface-exposed molecules detected by our enhanced gel microdroplet assay. In addition to underscoring the syphilis spirochete's remarkably poor surface antigenicity, our findings help to explain the complex and shifting balance between pathogen and host defenses that characterizes syphilitic infection.

Response of Reticulitermes hesperus (Isoptera: Rhinotermitidae) colonies to baiting with lufenuron in northern California.

The objective of this study was to evaluate lufenuron termite bait (1,500 ppm) for the elimination of colonies of Reticulitermes hesperus Banks (Isoptera: Rhinotermitidae). Dispersion of colonies in six baited and six unbaited sites near Placerville, CA, was determined by genetic (microsatellite) analyses. Twenty-one colonies of R. hesperus inhabited the six baited sites and eight colonies of R. hesperus occurred in the six unbaited sites. Five criteria provided a cause-and-effect link between the deployment of lufenuron termite bait and elimination of baited colonies: 1) association of foragers, as members of the same colony, in the independent monitoring stations and bait stations; 2) quantity of bait consumed; 3) abnormal physical appearance of foragers in bait stations; 4) disappearance of foragers from, and cessation of feeding in, independent monitoring stations visited by baited colonies; and 5) presence of foragers from, and continuation of feeding in, independent monitors visited by unbaited colonies. Baited colonies were devoid of foraging termites within a mean of 70.6 d (range, 37-93 d) of bait deployment. Colonies consumed a mean of 8.0 g of bait (range, 2.2-16.0 g). Wood consumption by baited and unbaited colonies was not significantly different during the 2 mo before baiting, 281.4 versus 590.5 mg/d per colony, respectively, nor during the 3 mo immediately after baiting, 112.5 versus 436.8 mg/d per colony, respectively. However, from 10 to 16 mo after baiting, wood consumption by baited colonies essentially ceased and was significantly less than the unbaited colonies, 7.9 versus 470.1 mg/d per colony, respectively.

Secondary syphilis in cali, Colombia: new concepts in disease pathogenesis.

Venereal syphilis is a multi-stage, sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum (Tp). Herein we describe a cohort of 57 patients (age 18-68 years) with secondary syphilis (SS) identified through a network of public sector primary health care providers in Cali, Colombia. To be eligible for participation, study subjects were required to have cutaneous lesions consistent with SS, a reactive Rapid Plasma Reagin test (RPR-titer > or = 1 : 4), and a confirmatory treponemal test (Fluorescent Treponemal Antibody Absorption test- FTA-ABS). Most subjects enrolled were women (64.9%), predominantly Afro-Colombian (38.6%) or mestizo (56.1%), and all were of low socio-economic status. Three (5.3%) subjects were newly diagnosed with HIV infection at study entry. The duration of signs and symptoms in most patients (53.6%) was less than 30 days; however, some patients reported being symptomatic for several months (range 5-240 days). The typical palmar and plantar exanthem of SS was the most common dermal manifestation (63%), followed by diffuse hypo- or hyperpigmented macules and papules on the trunk, abdomen and extremities. Three patients had patchy alopecia. Whole blood (WB) samples and punch biopsy material from a subset of SS patients were assayed for the presence of Tp DNA polymerase I gene (polA) target by real-time qualitative and quantitative PCR methods. Twelve (46%) of the 26 WB samples studied had quantifiable Tp DNA (ranging between 194.9 and 1954.2 Tp polA copies/ml blood) and seven (64%) were positive when WB DNA was extracted within 24 hours of collection. Tp DNA was also present in 8/12 (66%) skin biopsies available for testing. Strain typing analysis was attempted in all skin and WB samples with detectable Tp DNA. Using arp repeat size analysis and tpr RFLP patterns four different strain types were identified (14d, 16d, 13d and 22a). None of the WB samples had sufficient DNA for typing. The clinical and microbiologic observations presented herein, together with recent Cali syphilis seroprevalence data, provide additional evidence that venereal syphilis is highly endemic in this region of Colombia, thus underscoring the need for health care providers in the region to be acutely aware of the clinical manifestations of SS. This study also provides, for the first time, quantitative evidence that a significant proportion of untreated SS patients have substantial numbers of circulating spirochetes. How Tp is able to persist in the blood and skin of SS patients, despite the known presence of circulating treponemal opsonizing antibodies and the robust pro-inflammatory cellular immune responses characteristic of this stage of the disease, is not fully understood and requires further study.

The potential of backscattering interferometry as an in vitro clinical diagnostic tool for the serological diagnosis of infectious disease.

Backscattering interferometry enables the detection of syphilis antibody-antigen interactions in the presence of human serum, showing promise as a diagnostic tool for the serological diagnosis of infectious disease with potentially quantitative capabilities.

Broad specificity AhpC-like peroxiredoxin and its thioredoxin reductant in the sparse antioxidant defense system of Treponema pallidum.

Little is known about the mechanisms by which Treponema pallidum (Tp), the causative agent of syphilis, copes with oxidative stress as it establishes persistent infection within its obligate human host. The Tp genomic sequence indicates that the bacterium's antioxidant defenses do not include glutathione and are limited to just a few proteins, with only one, TP0509, offering direct defense against peroxides. Although this Tp peroxiredoxin (Prx) closely resembles AhpC-like Prxs, Tp lacks AhpF, the typical reductant for such enzymes. Functionally, TpAhpC resembles largely eukaryotic, nonAhpC typical 2-Cys Prx proteins in using thioredoxin (Trx, TP0919) as an efficient electron donor and exhibiting broad specificity toward hydroperoxide substrates. Unlike many of the eukaryotic Prxs, however, TpAhpC is relatively resistant to inactivation during turnover with hydroperoxide substrates. As is often observed in typical 2-Cys Prxs, TpAhpC undergoes redox-sensitive oligomer formation. Quantitative immunoblotting revealed that TpTrx and TpAhpC are present at very high levels (over 100 and 300 microM, respectively) in treponemes infecting rabbit testes; their redox potentials, at -242 +/- 1 and -192 +/- 2 mV, respectively, are consistent with the role of TpTrx as the cellular reductant of TpAhpC. Transcriptional analysis of select antioxidant genes confirmed the presence of high mRNA levels for ahpC and trx which diminish greatly when spirochetes replicate under in vitro growth conditions. Thus, T. pallidum has evolved an extraordinarily robust, broad-spectrum AhpC as its sole mechanism for peroxide defense to combat this significant threat to treponemal growth and survival during infection.

Cryo-electron tomography elucidates the molecular architecture of Treponema pallidum, the syphilis spirochete.

Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure's fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly parallel to the flagellar filaments along the inner surface of the cytoplasmic membrane (CM). Motile treponemes stably attached to rabbit epithelial cells predominantly via their tips. CET revealed that T. pallidum cell ends have a complex morphology and assume at least four distinct morphotypes. Images of dividing treponemes and organisms shedding cell envelope-derived blebs provided evidence for the spirochete's complex membrane biology. In the regions without flagellar filaments, peptidoglycan (PG) was visualized as a thin layer that divided the periplasmic space into zones of higher and lower electron densities adjacent to the CM and OM, respectively. Flagellar filaments were observed overlying the PG layer, while image modeling placed the PG-basal body contact site in the vicinity of the stator-P-collar junction. Bioinformatics and homology modeling indicated that the MotB proteins of T. pallidum, Treponema denticola, and Borrelia burgdorferi have membrane topologies and PG binding sites highly similar to those of their well-characterized Escherichia coli and Helicobacter pylori orthologs. Collectively, our results help to clarify fundamental differences in cell envelope ultrastructure between spirochetes and gram-negative bacteria. They also confirm that PG stabilizes the flagellar motor and enable us to propose that in most spirochetes motility results from rotation of the flagellar filaments against the PG.

The general transition metal (Tro) and Zn2+ (Znu) transporters in Treponema pallidum: analysis of metal specificities and expression profiles.

Acquisition of transition metals is central to the struggle between a bacterial pathogen and its mammalian host. Previous studies demonstrated that Treponema pallidum encodes a cluster-9 (C9) ABC transporter (troABCD) whose solute-binding protein component (TroA) ligands Zn(2+) and Mn(2+) with essentially equal affinities. Bioinformatic analysis revealed that T. pallidum encodes an additional C9 transporter (tp0034-36) orthologous to Zn(2+)-uptake (Znu) systems in other bacteria; the binding protein component, ZnuA, contains a His-rich tract characteristic of C9 Zn(2+)-binding proteins. Metal analysis and metal-reconstitution studies demonstrated that ZnuA is a Zn(2+)-binding protein; parallel studies confirmed that TroA binds Zn(2+), Mn(2+) and Fe. Circular dichroism showed that ZnuA, but not TroA, undergoes conformational changes in the presence of Zn(2+). Using isothermal titration calorimetry (ITC), we demonstrated that TroA binds Zn(2+) and Mn(2+) with affinities approximately 100-fold greater than those previously reported. ITC analysis revealed that ZnuA contains multiple Zn(2+)-binding sites, two of which are high-affinity and presumed to be located within the binding pocket and His-rich loop. Quantitative reverse transcription polymerase chain reaction of tro and znu transcripts combined with immunoblot analysis of TroA and ZnuA confirmed that both transporters are simultaneously expressed in T. pallidum and that TroA is expressed at much greater levels than ZnuA. Collectively, our findings indicate that T. pallidum procures transition metals via the concerted utilization of its general metal (Tro) and Zn(2+) (Znu) transporters. Sequestration of periplasmic Zn(2+) by ZnuA may free up TroA binding capacity for the importation of Fe and Mn(2+).

Diagnosis of gastric syphilis by direct immunofluorescence staining and real-time PCR testing.

We report on a case of gastric syphilis in a patient with chronic dyspepsia. The diagnosis was established by serology and the demonstration of spirochetes in diffusely inflammed gastric mucosa by staining with a fluorescent monoclonal antibody specific for pathogenic treponemes and by the detection of specific treponemal DNA sequences by a real-time PCR.

Eggshell composition of squamate reptiles: relationship between eggshell permeability and amino acid distribution.

Most snakes and lizards produce eggs with flexible shells that interact with the environment to maintain water balance. Geckos produce rigid eggshells that are independent of an external source of water and can be oviposited in more open, dryer locations. In this study, we analyzed and compared the amino acid composition of 24 lizard species, six snake species, and four outgroups (including avian and reptilian elastin and chicken eggshell). Rigid Gecko eggshells had significantly lower levels of seven of the 17 amino acids evaluated. Multivariate analysis showed that proline was the most important amino acid in distinguishing between these two groups of eggshells, occurring at significantly higher levels in flexible eggshells. High levels of proline have also been observed in the eggshells of other species. Proline and other amino acids are associated with the alleviation of water and salt stress in plants.

TP0453, a concealed outer membrane protein of Treponema pallidum, enhances membrane permeability.

The outer membrane of Treponema pallidum, the non-cultivable agent of venereal syphilis, contains a paucity of protein(s) which has yet to be definitively identified. In contrast, the outer membranes of gram-negative bacteria contain abundant immunogenic membrane-spanning beta-barrel proteins mainly involved in nutrient transport. The absence of orthologs of gram-negative porins and outer membrane nutrient-specific transporters in the T. pallidum genome predicts that nutrient transport across the outer membrane must differ fundamentally in T. pallidum and gram-negative bacteria. Here we describe a T. pallidum outer membrane protein (TP0453) that, in contrast to all integral outer membrane proteins of known structure, lacks extensive beta-sheet structure and does not traverse the outer membrane to become surface exposed. TP0453 is a lipoprotein with an amphiphilic polypeptide containing multiple membrane-inserting, amphipathic alpha-helices. Insertion of the recombinant, non-lipidated protein into artificial membranes results in bilayer destabilization and enhanced permeability. Our findings lead us to hypothesize that TP0453 is a novel type of bacterial outer membrane protein which may render the T. pallidum outer membrane permeable to nutrients while remaining inaccessible to antibody.

A monoclonal antibody that conveys in vitro killing and partial protection in experimental syphilis binds a phosphorylcholine surface epitope of Treponema pallidum.

Immunization with purified Treponema pallidum outer membrane vesicles (OMV) has previously resulted in high-titer complement-dependent serum bactericidal activity. In this study, OMV immunization resulted in the isolation of a monoclonal antibody, M131, with complement-dependent killing activity. Passive immunization of rabbits with M131 administered intravenously conferred significant immunity demonstrated by the failure of syphilitic lesions to appear at 29% of intradermal challenge sites (7/24) and a mean delay of approximately 8 days to lesion appearance at the remaining sites (17/24). M131 not only bound to OMV and to the surfaces of intact motile T. pallidum cells but also bound to organisms whose outer membranes were removed, indicating both surface and subsurface locations for the killing target. This target was determined to be a T. pallidum lipid. Lipid extracted from T. pallidum and made into liposomes bound M131. Reverse-phase high-pressure liquid chromatography separation and fraction collection mass spectrometry (LC-MS+) of T. pallidum lipid showed that the target of M131 was phosphorylcholine. M131 binding required both liposome formation and a critical concentration of phospholipid containing phosphorylcholine, suggesting that the epitope has both a conformational and a compositional requirement. M131 did not react with red blood cells, which have phosphorylcholine-containing lipids in their exterior membrane leaflets, or with Venereal Disease Research Laboratory antigen that also contains phosphorylcholine, further indicating the specificity of M131. This is the first physical demonstration of an antigen on the T. pallidum surface and indication that such a surface antigen can be a target of immunity.

Susceptibility of Treponema pallidum to host-derived antimicrobial peptides.

LL-37 displays potent broad-spectrum activity against a number of pathogenic bacteria and is the only cathelicidin thus far identified in humans. In this study, we examined the capacity of human LL-37 and the similar CAP-18-derived peptide from rabbits to exert antimicrobial activity against the causative agent of syphilis, Treponema pallidum. We found that both peptides, as well as a truncated version of human LL-37 that contains its bactericidal domain, could exert rapid, but salt-sensitive antimicrobial activity against T. pallidum. Infectivity of T. pallidum in a rabbit model could effectively be blocked with the synthetic truncated LL-37-derived peptide WS22-N-amide.

Insertion of fluorescent fatty acid probes into the outer membranes of the pathogenic spirochaetes Treponema pallidum and Borrelia burgdorferi.

The authors examined the ability of octadecanoyl (C(18)), hexadecanoyl (C(16)) and dodecanoyl (C(12)) fatty acid (FA) conjugates of 5-aminofluorescein (OAF, HAF and DAF, respectively) to insert into the outer membranes (OMs) of Treponema pallidum, Borrelia burgdorferi and Escherichia coli. Biophysical studies have demonstrated that these compounds stably insert into phospholipid bilayers with the acyl chain within the hydrophobic interior of the apical leaflet and the hydrophilic fluorescein moiety near the phospholipid head groups. Consistent with the known poor intrinsic permeability of the E. coli OM to hydrophobic compounds and surfactants, E. coli was not labelled with any of the FA probes. OAF inserted more readily into OMs of B. burgdorferi than into those of T. pallidum, although both organisms were completely labelled at concentrations at or below 2 microg ml(-1). Intact spirochaetes were labelled with OAF but not with antibodies against known periplasmic antigens, thereby confirming that the probe interacted exclusively with the spirochaetal OMs. Separate experiments in which organisms were cooled to 4 degrees C (i.e. below the OM phase-transition temperatures) indicated that labelling with OAF was due to insertion of the probe into the OMs. B. burgdorferi, but not T. pallidum, was labelled by relatively high concentrations of HAF and DAF. Taken as a whole, these findings support the prediction that the lack of lipopolysaccharide renders T. pallidum and B. burgdorferi OMs markedly more permeable to lipophilic compounds than their Gram-negative bacterial counterparts. The data also raise the intriguing possibility that these two pathogenic spirochaetes obtain long-chain FAs, nutrients they are unable to synthesize, by direct permeation of their OMs.