Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands

BACKGROUND : The Ehrlichia are obligate intracellular Gram-negative tick-borne bacteria that are important human and animal pathogens. There is a need for assays to rapidly and reliably detect and differentiate the five generally recognized species into groups in a single reaction: E. canis, E. chaffeensis, E. ewingii, E. muris and E. ruminantium. METHODS : We developed primers and probes against the 16S rRNA gene to enable us to reliably detect the five major Ehrlichia spp. in a single FRET-qPCR. We tested the Ehrlichia FRET-qPCR on reference strains and on DNA from the blood of domestic ruminants from five Caribbean islands. The Ehrlichia present were determined using melting point analysis and by sequencing the Ehrlichia FRET-qPCR products as well as those of a nested PCR against the citrate synthase gene (gltA). RESULTS : Our Ehrlichia FRET-qPCR was negative for the closely related Anaplasma marginale and A. phagocytophilum but gave positive reactions with reference strains of the most generally recognized species and with other less characterized Ehrlichia of domestic ruminants, mainly E. ovina, the Panola Mountain Ehrlichia, and Ehrlichia sp. BOV2010. Melting point analysis revealed 4 distinct groups: E. ruminantium (Tm ~55.8 °C); E. chaffeensis and E. ewingii (Tm ~57.7 °C); E. canis, E. muris, E. ovina and Ehrlichia sp. BOV 2010 (Tm ~62.0 °C); and the Panola Mountain Ehrlichia (Tm ~65.5 °C). The detection limit of the FRET-qPCR was ~ 5 gene copies in a reaction and the sequences of the FRET-qPCR products were as expected. With DNA from domestic ruminants from the Caribbean we found 12.2 % (134/1,101) positive: cattle (76/385; 19.7 %), sheep (45/340; 13.2 %) and goats (13/376; 3.5 %). Melting point analysis and sequencing of the FRET-qPCR and nested PCR gltA products showed the Ehrlichia we detected were E. canis or very closely related organisms. CONCLUSIONS : In a single reaction, our Ehrlichia FRET-qPCR can detect the Ehrlichia spp. we studied and differentiate them into four groups. Domestic ruminants in the Caribbean are not uncommonly exposed to Ehrlichia, possibly E. canis or very closely related organisms.Grants from the National Natural Science Foundation of China (NO: 31272575), the Priority Academic Program Development of Jiangsu Higher Education Institutions, Yangzhou, Jiangsu, P. R. China and the Ross University School of Veterinary Medicine.http://www.parasitesandvectors.comam201

Human Infection from Avian-like Influenza A (H1N1) Viruses in Pigs, China

In investigating influenza in an immunodeficient child in China, in December 2010, we found that the influenza virus showed high sequence identity to that of swine. Serologic evidence indicated that viral persistence in pigs was the source of infection. Continued surveillance of pigs and systemic analysis of swine influenza isolates are needed

Molecular Detection of Theileria spp. in Livestock on Five Caribbean Islands

Theileria spp. are tick-transmitted, intracellular apicomplexan protozoan parasites infecting a wide range of animals. As there is very limited information on the prevalence of Theileria spp. in the Caribbean we used the recently described genus-specific pan-Theileria FRET-qPCR to identify infected animals in the region and a standard 18S rRNA gene PCR and sequencing to determine the species involved. We found Theileria spp. in 9% of the convenience samples of animals (n=752) studied from five Caribbean islands. Donkeys (20.0%: 5/25) were most commonly infected, followed by sheep (17.4%, 25/144), cattle (6.8%; 22/325), goats (5.0%; 12/238), and horses (5.0%; 1/20). Six species of Theileria were identified: T. equi (donkeys, cattle, goats, and sheep), Theileria sp. OT3 (sheep and goats), Theileria sp. NG-2013a (cattle), Theileria sp. YW-2014 (donkeys), Theileria sp. B15a (goats), and Babesia vulpes or a closely related organism (sheep and goats). Only T. equi has been previously reported in the Caribbean. Our findings expand the known host ranges of Theileria spp. and the known distribution of the organisms around the world

Biomass‐based materials for advanced supercapacitor: principles, progress, and perspectives

Abstract Supercapacitors exhibit considerable potential as energy storage devices due to their high power density, fast charging and discharging abilities, long cycle life, and eco‐friendliness. With the increasing environmental concerns associated with synthetic compounds, the use of environment friendly biopolymers to replace conventional petroleum‐based materials has been widely studied. Biomass‐based materials are biodegradable, renewable, environment friendly and non‐toxic. The unique hierarchical nanostructure, excellent mechanical properties and hydrophilicity allow them to be used to create functional conductive materials with precisely controlled structures and different properties. In this review, the latest development of biomass‐based supercapacitor materials is reviewed and discussed. This paper describes the physical and chemical properties of various biopolymers and their impact on supercapacitors, as well as the classification and basic principles of supercapacitors. Then, a comprehensive discussion is presented on the utilization of biomass‐based materials in supercapacitors and their recent applications across a range of supercapacitor devices. Finally, an overview of the future prospects and challenges pertaining to the utilization of biomass‐based materials in supercapacitors is provided

Cellulose-based materials for carbon capture and conversion

With the worldwide attention on decreasing carbon dioxide (CO2) emissions, carbon capture and storage (CCS) is utilized to stop CO2 from escaping into the atmosphere. Selecting appropriate materials is crucial for the establishment of a dependable and secure carbon capture and storage technology infrastructure. The natural cellulose materials possess inherent characteristics such as eco-friendliness, high utility, remarkable physical and mechanical properties, which make them particularly available for CCS. In the field of carbon capture, utilization and sequestration technologies, cellulose-based materials can be used as adsorbents for CO2 capture and as catalyst carriers for CO2 conversion. Firstly, the application of cellulose-based materials in carbon capture and conversion is introduced. Secondly, the research progress of cellulose-based films, aerogels and solid adsorbents in the field of carbon capture are summarized. Subsequently, the research mechanisms of cellulose-based materials for the conversion of CO2 to methanol format, carbonate and CO are also described in detail. Finally, the challenges and the key problems of cellulose-based materials in carbon capture and utilization are described

Diagnosis of canine leptospirosis by a highly sensitive FRET-PCR targeting the lig genes.

Canine leptospirosis is underdiagnosed due to its wide spectrum of clinical presentations and the lack of a rapid and sensitive test for the accurate diagnosis of acute and chronic infections. In this study, we developed a highly sensitive and specific fluorescence resonance energy transfer (FRET)-PCR to detect common pathogenic leptospires in dogs, including Leptospira interrogans serovars Autumnalis, Canicola, Copenhageni (Icterohaemorrhagiae serogroup) and Pomona, and Leptospira kirschneri serovar Grippotyphosa. This PCR targets the lig genes, exclusively found in the pathogenic Leptospira species but not in saprophytic species (L. biflexa). A robust, high-stringency step-down real-time platform was coupled to the highly specific detection of leptospiral DNA by fluorescently labeled FRET probes. This enabled the detection of a single copy of the lig gene in a PCR containing DNA from up to 50 µL canine blood or 400 µL urine. Sensitivity determination by use of limiting serial dilutions of extracted leptospiral DNA indicated that the lig FRET-PCR we established was almost 100-fold more sensitive than the widely accepted lipL32 SYBR assay and 10-fold more sensitive than a 16S rRNA TaqMan assay. Application of this method to 207 dogs with potential leptospiral infection enabled us to diagnose three cases of canine leptospirosis characterized by low amounts of leptospiral DNA in body fluids. Detection of canine leptospirosis with the lig FRET-PCR was more sensitive with the lig FRET-PCR than with the 16S rRNA TaqMan PCR, which detected only 2 of the 3 cases, and the lipL32 SYBR PCR, which detected none of the 3 dogs with leptospirosis

Molecular Detection of Theileria spp. in Livestock on Five Caribbean Islands

Theileria spp. are tick-transmitted, intracellular apicomplexan protozoan parasites infecting a wide range of animals. As there is very limited information on the prevalence of Theileria spp. in the Caribbean we used the recently described genus-specific panTheileria FRET-qPCR to identify infected animals in the region and a standard 18S rRNA gene PCR and sequencing to determine the species involved. We found Theileria spp. in 9% of the convenience samples of animals ( = 752) studied from five Caribbean islands. Donkeys (20.0%: 5/25) were most commonly infected, followed by sheep (17.4%, 25/144), cattle (6.8%; 22/325), goats (5.0%; 12/238), and horses (5.0%; 1/20). Six species of Theileria were identified: T. equi (donkeys, cattle, goats, and sheep), Theileria sp. OT3 (sheep and goats), Theileria sp. NG-2013a (cattle), Theileria sp. YW-2014 (donkeys), Theileria sp. B15a (goats), and Babesia vulpes or a closely related organism (sheep and goats). Only T. equi has been previously reported in the Caribbean. Our findings expand the known host ranges of Theileria spp. and the known distribution of the organisms around the world

Development of a Whole-Cell Biocatalyst/Biosensor by Display of Multiple Heterologous Proteins on the Escherichia coli Cell Surface for the Detoxification and Detection of Organophosphates

This paper reports the codisplay of organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH)–green fluorescent protein (GFP) fusion on the cell surface of Escherichia coli using the truncated ice nucleation protein (INPNC) and Lpp–OmpA as the anchoring motifs. The surface localization of both OPH and MPH–GFP was demonstrated by cell fractionation, Western blot analysis, protease accessibility experiment, and immunofluorescence microscopy. Anchorage of the foreign proteins on the outer membrane neither inhibits cell growth nor affects cell viability. The recombinant strain can be used as a whole-cell biocatalyst and showed a broader substrate range than strains expressing either OPH or MPH. A mixture of six organophosphorus pesticides (OPs) (0.2 mM each) could be degraded completely within 5 h. The broader substrate specificity in combination with the rapid degradation rate makes the recombinant strain a promising candidate for detoxification of OPs. The fluorescence of surface-displayed GFP is very sensitive to environmental pH change. Because hydrolysis of OPs by OPH or MPH generates protons, the recombinant <i>E. coli</i> could be used as a whole-cell biosensor for the rapid detection of OPs by evaluating fluorescence changes as a function of OP concentrations