Development of PCR/dot blot assay for specific detection and differentiation of taeniid cestode eggs in canids

We report the development of a colourimetric PCR/dot blot assay targeting the mitochondrial gene NADH dehydrogenase subunit 1 (nad1) for differential diagnosis of taeniid eggs. Partial sequences of the cestode nad1 gene were aligned and new primers were designed based on conserved regions. Species-specific oligonucleotide probes (S-SONP) for canine taeniid cestodes were then designed manually based on the variable region between the conserved primers. Specifically, S-SONP were designed for the Taenia crassiceps, T. hydatigena, T. multiceps, T. ovis, T. taeniaeformis, Echinococcus granulosus (genotype 1), E. multilocularis and E. vogeli. Each probe showed high specificity as no cross-hybridisation with any amplified nad1 fragment was observed. We evaluated the assay using 49 taeniid egg-positive samples collected from dogs in Zambia. DNA from 5 to 10 eggs was extracted in each sample. Using the PCR/dot blot assay, the probes successfully detected PCR products from T. hydatigena in 42 samples, T. multiceps in 3 samples, and both species (mixed infection) in the remaining 4 samples. The results indicate that the PCR/dot blot assay is a reliable alternative for differential diagnosis of taeniid eggs in faecal samples.     

Serologic and molecular diagnosis of zoonotic larval cestode infections

Neurocysticercosis, cystic echinococcosis and alveolar echinococcosis are the three major zoonotic larval cestodiases worldwide and threatening human life. Early therapeutic treatment based on early differential diagnosis at the early stage of infection and epidemiological surveillance with the expectation for control and prevention are the keys for improvement of quality of human life. Recent advances in (1). differential serodiagnosis of these three diseases, (2). molecular identification of human taeniid species and (3). polymorphism of mitochodrial DNA highly informative for future molecular epidemiology are reviewed in this article.     

Molecular and genetic characterisation of the host-protective oncosphere antigens of taeniid cestode parasites

Highly effective recombinant vaccines have been developed against Taenia ovis infection in sheep, Taenia saginata infection in cattle, Taenia solium infection in pigs, Echinococcus granulosus and Echinococcus multilocularis infections in a variety of intermediate host species. These vaccines have been based on the identification and expression in Escherichia coli of antigens derived from the oncosphere life cycle stage, contained within the parasites' eggs. Investigation of the molecular aspects of these proteins and the genes encoding them have revealed a number of common features, including the presence of a predicted secretory signal sequence, and one or two copies of a fibronectin type III domain, each encoded by separate exons within the associated gene. Evidence has been obtained to confirm glycosylation of some of these antigens. Ongoing investigations will shed light on the biological roles played by the proteins within the parasites and the mechanism by which they make the parasites vulnerable to vaccine-induced immune responses.     

Echinococcus shiquicus n. sp., a taeniid cestode from Tibetan fox and plateau pika in China

The taeniid cestode Echinococcus shiquicus n. sp. was found from the Tibetan fox Vulpes ferrilata and the plateau pika Ochotona curzoniae in the Qinghai-Tibet plateau region of China. In the adult stage, E. shiquicus from the foxes is morphologically similar to Echinococcus multilocularis. However, the new species is differentiated by its smaller rostellar hooks, fewer segments, distinct position of genital pore in the mature segment and fewer eggs in the gravid segment. Hydatid cysts of E. shiquicus found in the livers from the pikas were essentially unilocular but an oligovesicular cyst was also found. The data of mitochondrial and nuclear DNA sequences proved E. shiquicus to be a valid taxon.     

Evaluation of the Vitek Immunodiagnostic Assay System (VIDAS) for the detection of Salmonella in foods

A Salmonella Assay using the Vitek Immunodiagnostic Assay System (VIDAS) was compared with a conventional cultural method (CCM) for the detection of salmonellas in 141 samples of artificially and naturally contaminated foods. There was an overall agreement of 92.9% between the methods. The productivity of the VIDAS Salmonella Assay (VSA) was not improved using an alternative enrichment protocol for the detection of Salmonella in 12 raw meat samples.
The sensitivity and specificity of the VSA was assessed using pure cultures of salmonellas and non-salmonellas. The detection limit was 1.8 times 10⁶ salmonellas ml^-1 in M-broth and some Citrobacter freundii strains gave false-positive results.
Using an immunomagnetic separation (IMS) technique and an abbreviated cultural enrichment, the VSA results could be obtained a day earlier than the standard VSA method.     

Immunodiagnostic Assays for the Investigation of Fungal Outbreaks

Mycopathologia - Fungal pathogens can affect humans, animals, and plants, and they can be found in the environment or as part of the host microbiome. Fungal diseases present a broad clinical...     

Multiple infections: relatedness and time between infections affect the establishment and growth of the cestode Schistocephalus solidus in its stickleback host

We studied experimental double infections of the cestode Schistocephalus solidus in its stickleback host. In particular, we were interested in how two important components of the cestode's transmission success-establishment and growth within the fish host-were affected by the relatedness of the two parasites in a double exposure and by the timing of the two exposures, that is, whether they occurred simultaneously or sequentially. We found that male sticklebacks more often became infected (singly or doubly) if the two cestodes in the exposures were related, whereas female sticklebacks were more easily infected (singly or doubly) when exposed to two unrelated cestodes. Irrespective of the fish's gender, successful infections more often contained both cestodes when they were related. In sequential exposures with related as well as unrelated cestodes, the cestode in the later exposure survived better and also grew larger than the cestode from the first exposure, despite being one week younger. Our results emphasize that within-host dynamics and factors acting at this level can play an important role in determining a parasite's transmission success.     

Therapeutic approaches for combating Pseudomonas aeruginosa infections

Pseudomonas aeruginosa is an opportunistic pathogen responsible for acute hospital-acquired infections. This review described various therapeutic approaches to treat infections caused by P. aeruginosa, including conventional therapy, novel antibiotic treatments and treatments other than antibiotics. Most of the developments are still in research that will provide novel treatment options in future.     

Proteomic Selection of Immunodiagnostic Antigens for Human African Trypanosomiasis and Generation of a Prototype Lateral Flow Immunodiagnostic Device

Background

The diagnosis of Human African Trypanosomiasis relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). While this test is successful, it is acknowledged that there may be room for improvement. Our aim was to develop a prototype lateral flow test based on the detection of antibodies to trypanosome antigens.

Methodology/Principal Findings

We took a non-biased approach to identify potential immunodiagnostic parasite protein antigens. The IgG fractions from the sera from Trypanosoma brucei gambiense infected and control patients were isolated using protein-G affinity chromatography and then immobilized on Sepharose beads. The IgG-beads were incubated with detergent lysates of trypanosomes and those proteins that bound were identified by mass spectrometry-based proteomic methods. This approach provided a list of twenty-four trypanosome proteins that selectively bound to the infection IgG fraction and that might, therefore, be considered as immunodiagnostic antigens. We selected four antigens from this list (ISG64, ISG65, ISG75 and GRESAG4) and performed protein expression trials in E. coli with twelve constructs. Seven soluble recombinant protein products (three for ISG64, two for ISG65 and one each for ISG75 and GRESAG4) were obtained and assessed for their immunodiagnostic potential by ELISA using individual and/or pooled patient sera. The ISG65 and ISG64 construct ELISAs performed well with respect to detecting T. b. gambiense infections, though less well for detecting T. b. rhodesiense infections, and the best performing ISG65 construct was used to develop a prototype lateral flow diagnostic device.

Conclusions/Significance

Using a panel of eighty randomized T. b. gambiense infection and control sera, the prototype showed reasonable sensitivity (88%) and specificity (93%) using visual readout in detecting T. b. gambiense infections. These results provide encouragement to further develop and optimize the lateral flow device for clinical use.     

New DNA-based PCR approaches for rapid real-time detection and prevention of group B streptococcal infections in newborns and pregnant women

Group B streptococci (GBS) are an important cause of neonatal sepsis and meningitis, and maternal infection. Although the pathogenesis of GBS infection is not well understood, several virulence factors have been identified. Two prevention strategies have been proposed: chemoprophylaxis and immunoprophylaxis. Implementation of selective intrapartum chemoprophylaxis on the basis of either screening or risk assessment has led to a substantial decrease in the morbidity and mortality of GBS disease in both mothers and infants. Penicillin remains the antibiotic of choice with no reported resistant GBS so far, whereas resistance of 10-20% of GBS to erythromycin and clindamycin has been reported in North America. Chemoprophylaxis based on screening requires optimal detection methods for GBS, which involve selective broth culture of combined vaginal and anal samples. Other conventional methods are useful for rapid identification of heavily colonised women, but are unreliable for the detection of light GBS colonisation because of poor sensitivity. GBS-specific polymerase chain reaction (PCR) assays using real-time PCR coupled with fluorescence-labelling technology offer powerful tools for sensitive and specific, yet rapid (less than 1 h), detection of GBS directly from clinical specimens at the time of delivery. The application of these assays to the current prevention strategies will simplify the prevention practice and rationalise the use of antibiotics. Immunoprophylaxis relies on the development of new vaccines against GBS, and active research is being conducted in this area.     

MicroRNA定量检测方法的研究进展

MicroRNA是一类内源性的非编码小分子RNA, 通过下调蛋白编码基因的表达而对不同的细胞发育过程起到重要的调控作用。分析组织或细胞样本中microRNA的表达可为研究这类分子的生物学功能提供重要的信息。近年来, 研究者发展了许多方法检测不同的生理和病理学过程中microRNA的表达差异, 并发现microRNA的异常表达与癌症、神经紊乱和心脏疾病等的发生相关。文章系统地介绍了最新发展的microRNA定量检测方法, 详细阐述了基于探针杂交技术的Northern blotting法、微阵列芯片法、纳米金标记法、桥连同位素标记法, 以及基于扩增技术的定量PCR检测法、滚环扩增法、引物入侵法和新一代大规模高通量测序法等, 并对这些方法的优缺点进行了分析比较。     

New approaches in vaccine development for parasitic infections

Vaccines have had a tremendous impact on the control of infectious diseases. Not only are vaccines potentially the least expensive mechanism to combat infectious diseases, under optimal conditions, widespread vaccination can result in disease eradication - as in the case of smallpox. Despite this great potential, vaccines have had little impact on human parasitic infections. The reasons for this are many - these eukaryotic pathogens are genetically and biologically complex organisms, some with elaborate life cycles and well-honed immune evasion mechanisms. Additionally, our understanding of the mechanisms of immune control of many parasitic infections -- of what constitutes an effective immune response and of how to induce high-quality immunological memory -- is not fully developed. This review attempts to highlight recent advances that could impact vaccine discovery and development in parasitic infections and proposes areas where future studies may lead to breakthroughs in vaccines for the agents of parasitic diseases. There are several other recent reviews highlighting the results of vaccine trials, specifically in the malaria field.     

Proteogenomic approaches for the molecular characterization of natural microbial communities

At the present time we know little about how microbial communities function in their natural habitats. For example, how do microorganisms interact with each other and their physical and chemical surroundings and respond to environmental perturbations? We might begin to answer these questions if we could monitor the ways in which metabolic roles are partitioned amongst members as microbial communities assemble, determine how resources such as carbon, nitrogen, and energy are allocated into metabolic pathways, and understand the mechanisms by which organisms and communities respond to changes in their surroundings. Because many organisms cannot be cultivated, and given that the metabolisms of those growing in monoculture are likely to differ from those of organisms growing as part of consortia, it is vital to develop methods to study microbial communities in situ. Chemoautotrophic biofilms growing in mine tunnels hundreds of meters underground drive pyrite (FeS(2)) dissolution and acid and metal release, creating habitats that select for a small number of organism types. The geochemical and microbial simplicity of these systems, the significant biomass, and clearly defined biological-inorganic feedbacks make these ecosystem microcosms ideal for development of methods for the study of uncultivated microbial consortia. Our approach begins with the acquisition of genomic data from biofilms that are sampled over time and in different growth conditions. We have demonstrated that it is possible to assemble shotgun sequence data to reveal the gene complement of the dominant community members and to use these data to confidently identify a significant fraction of proteins from the dominant organisms by mass spectrometry (MS)-based proteomics. However, there are technical obstacles currently restricting this type of "proteogenomic" analysis. Composite genomic sequences assembled from environmental data from natural microbial communities do not capture the full range of genetic potential of the associated populations. Thus, it is necessary to develop bioinformatics approaches to generate relatively comprehensive gene inventories for each organism type. These inventories are critical for expression and functional analyses. In proteomic studies, for example, peptides that differ from those predicted from gene sequences can be measured, but they generally cannot be identified by database matching, even if the difference is only a single amino acid residue. Furthermore, many of the identified proteins have no known function. We propose that these challenges can be addressed by development of proteogenomic, biochemical, and geochemical methods that will be initially deployed in a simple, natural model ecosystem. The resulting approach should be broadly applicable and will enhance the utility and significance of genomic data from isolates and consortia for study of organisms in many habitats. Solutions draining pyrite-rich deposits are referred to as acid mine drainage (AMD). AMD is a very prevalent, international environmental problem associated with energy and metal resources. The biological-mineralogical interactions that define these systems can be harnessed for energy-efficient metal recovery and removal of sulfur from coal. The detailed understanding of microbial ecology and ecosystem dynamics resulting from the proposed work will provide a scientific foundation for dealing with the environmental challenges and technological opportunities, and yield new methods for analysis of more complex natural communities.     

New approaches in the treatment of bacterial infections

Recently, several new drugs for the treatment of bacterial infections have been developed. Quinupristin/dalfopristin, moxifloxacin and gatifloxacin have been approved throughout the world for clinical use. Levofloxacin has been approved for the treatment of community-acquired pneumonia caused by penicillin-resistant Streptococcus pnuemoniae. The Food and Drug Administration has approved linezolid for clinical use, and new drug applications for gemifloxacin and telithromycin were filed. Other new targets have surfaced in the quest for novel antibacterial agents.