Molecular and immunological characterisation of tropomyosin from Anisakis pegreffii.

Tropomyosin (TM) is a major allergen in shellfish, known to cross-react with mite, cockroach and/or some roundworm (nematode) TM. In this study, we aimed to express and purify TM from the parasitic nematode Anisakis pegreffii and also to characterise its cross-reactivity with TM from shellfish. A. pegreffii was isolated from the flathead tiger fish (Neoplatycephalus richardsoni) and characterised using single-strand conformation polymorphism (SSCP)-based sequencing of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA. The recombinant tropomyosin (rTM) of A. pegreffii was expressed, purified and confirmed by immunohistochemistry, sequencing and LC-MS/MS analyses. Immunohistochemistry showed the muscle and the base layer of the third-stage larvae (L3) of A. pegreffii as the location of TM in A. pegreffii. The molecular relationship of TM of A. pegreffii with homologs from other nematodes and crustaceans was inferred from phylogenetic analysis. Immunogenicity of TM from A. pegreffii was tested by immunoblotting, which showed that rTM from A. pegreffii binds to IgE from sera of patients with allergy to crustaceans. Immunoblotting also showed that the anti-TM monoclonal antibody (MAb) did not recognise rTM from A. pegreffii. The rTM from A. pegreffii was, however, recognised by anti-TM polyclonal antibodies (PAbs) as well as anti-crustacean polyclonal antibodies (PAbs). The detection of specific serum IgE antibody against parasite TM has been proposed as a useful approach for the diagnosis of parasite-induced allergy. The findings of this study merit further exploration of the cross-reactive allergenic proteins of Anisakis for improved, future diagnosis of allergenic diseases.

A global virulence regulator in Acinetobacter baumannii and its control of the phenylacetic acid catabolic pathway.

BACKGROUND: Acinetobacter baumannii is one of the most notorious hospital-acquired pathogens, and novel treatment strategies are desperately required. Two-component regulatory systems represent potential therapeutic targets as they mediate microorganism adaptation to changing environments, often control virulence, and are specific to bacteria. Here we describe the first global virulence regulator in A. baumannii. METHODS AND RESULTS: Using transcriptional profiling and functional assays of a deletion mutant in the A. baumannii sensor kinase gene, A1S_0574 (termed as gacS), we show that this sensor kinase regulates key virulence characteristics, including pili synthesis, biofilms, and motility, resulting in virulence attenuation in a mammalian septicemia model. Notably, we also identified that GacS regulates an operon novel to A. baumannii (paa operon), which is responsible for the metabolism of aromatic compounds. Deletion of paaE (A1S_1340) confirmed the role of this operon in A. baumannii virulence. Finally, we identified the cognate response regulator (A1S_0236) for GacS and confirmed their interaction. A1S_0236 was shown to regulate 75% of the GacS transcriptome and the same virulence phenotypes. Overexpression of A1S_0236 restored virulence in the gacS mutant. CONCLUSIONS: Our study characterizes a global virulence regulator, which may provide an alternate therapeutic target, in one of the most troublesome hospital-acquired pathogens.

An Evaluation of Urease A Subunit Nanocapsules as a Vaccine in a Mouse Model of Helicobacter pylori Infection.

Using removable silica templates, protein nanocapsules comprising the A subunit of Helicobacter pylori urease (UreA) were synthesised. The templates were of two sizes, with solid core mesoporous shell (SC/MS) silica templates giving rise to nanocapsules of average diameter 510 nm and mesoporous (MS) silica templates giving rise to nanocapsules of average diameter 47 nm. Both were shown to be highly monodispersed and relatively homogenous in structure. Various combinations of the nanocapsules in formulation were assessed as vaccines in a mouse model of H. pylori infection. Immune responses were evaluated and protective efficacy assessed. It was demonstrated that vaccination of mice with the larger nanocapsules combined with an adjuvant was able to significantly reduce colonisation.

Anisakis Nematodes in Fish and Shellfish- from infection to allergies.

Anisakidosis is a zoonotic parasitosis induced by members of the family Anisakidae. The anisakid genera includes Anisakis, Pseudoterranova, Hysterothylacium and Contracaecum. The final definitive hosts of these nematodes are marine mammals with a complex life cycle. These nematode parasites use different crustaceans and fish species as intermediate or paratenic hosts and humans are accidental hosts. Human anisakiasis, the infections caused by members of the genus Anisakis, occurs, when seafoods, particularly fish, contaminated with the infective stage (third stage larvae [L3]) of this parasite, are consumed. Pseudoterranovosis, on the other hand is induced by members of the genus Pseudoterranova. These two genera of anisakids have been implicated in human disease globally. There is a rise in reports of gastro-intestinal infections accompanied by allergic reactions caused by Anisakis simplex and Anisakis pegreffii. This review provides an update on current knowledge on Anisakis as a food-borne parasite with special focus on the increasingly reported diversity of fish and crustacean hosts, allergens and immunological cross-reactivity with invertebrate proteins rendering this parasite a significant public health issue.

The Anisakis Transcriptome Provides a Resource for Fundamental and Applied Studies on Allergy-Causing Parasites.

BACKGROUND: Food-borne nematodes of the genus Anisakis are responsible for a wide range of illnesses (= anisakiasis), from self-limiting gastrointestinal forms to severe systemic allergic reactions, which are often misdiagnosed and under-reported. In order to enhance and refine current diagnostic tools for anisakiasis, knowledge of the whole spectrum of parasite molecules transcribed and expressed by this parasite, including those acting as potential allergens, is necessary. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we employ high-throughput (Illumina) sequencing and bioinformatics to characterise the transcriptomes of two Anisakis species, A. simplex and A. pegreffii, and utilize this resource to compile lists of potential allergens from these parasites. A total of ~65,000,000 reads were generated from cDNA libraries for each species, and assembled into ~34,000 transcripts (= Unigenes); ~18,000 peptides were predicted from each cDNA library and classified based on homology searches, protein motifs and gene ontology and biological pathway mapping. Using comparative analyses with sequence data available in public databases, 36 (A. simplex) and 29 (A. pegreffii) putative allergens were identified, including sequences encoding 'novel' Anisakis allergenic proteins (i.e. cyclophilins and ABA-1 domain containing proteins). CONCLUSIONS/SIGNIFICANCE: This study represents a first step towards providing the research community with a curated dataset to use as a molecular resource for future investigations of the biology of Anisakis, including molecules putatively acting as allergens, using functional genomics, proteomics and immunological tools. Ultimately, an improved knowledge of the biological functions of these molecules in the parasite, as well as of their immunogenic properties, will assist the development of comprehensive, reliable and robust diagnostic tools.

In vitro antimicrobial activity of crude extracts from plants Bryophyllum pinnatum and Kalanchoe crenata.

Extracts from the leaves of Bryophyllum pinnatum and Kalanchoe crenata were screened for their antimicrobial activities. Solvents used included water, methanol, and local solvents such as palmwine, local gin (Seaman's Schnapps 40% alcoholic drink,) and "omi ekan-ogi" (Sour water from 3 days fermented milled maize). Leaves were dried and powdered before being soaked in solvents for 3 days. Another traditional method of extraction by squeezing raw juice from the leaves was also employed. All extracts were lyophilized. These extracts were tested against some gram-negative organisms (Escherichia coli ATCC 25922, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella flexneri, Salmonella paratyphi, Citrobacter spp); gram-positive organisms Staphylococcus aureus ATCC 25213, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis) and a fungus (Candida albicans). Agar well diffusion and broth dilution methods were used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) at concentrations of 512 mg/ml to 4 mg/ml. All the organisms except Candida albicans were susceptible to the extracts obtained from the traditional method. The squeezed-leaf juice of Kalanchoe crenata was the most active one with MIC of 8 mg/ml against Pseudomonas aeruginosa, Klebsiella pneumoniae and Bacillus subtilis, 32 mg/ml against Shigella flexneri, 64 mg/ml against Escherichia coli and 128 mg/ml against the control strain Staphylococcus aureus while its MBC is 256 mg/ml against these organisms except Bacillus subtilis and Klebsiella pneumoniae. The gram-positive organisms were more sensitive to the methanol and local gin-extract of Bryophyllum pinnatum. Extracts from other solvents showed moderate to weak activity.

Evaluation of the antimicrobial properties of different parts of Citrus aurantifolia (lime fruit) as used locally.

We investigated the potency of Citrus aurantifolia (Lime fruit), against pathogens, in the different forms in which this fruit plant is used locally (juice of the fruit, burnt rind of the fruit commonly known as "epa-ijebu" in the Yoruba dialect) and the oil obtained from steam distillation of the fruit. The antimicrobial activity of "epa-ijebu" in different solvents was also compared. The solvents include palm-wine (a local alcoholic drink tapped from palm trees), Seaman's Schnapps 40% alcoholic drink, water, ethanol and fermented water from 3 days soaked milled maize known as "ekan-ogi" or "omidun" in the Yoruba dialect. Antimicrobial activity was carried out by the agar well diffusion. The clinical isolates used included Anaerobic facultative bacteria, namely: Staphylococcus aureus ATCC 25213, Staphylococcus aureus, Salmonella paratyphi, Shigella flexnerii, Streptococcus faecalis, Citrobacter spp, Serratia spp, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli ATCC 25922, and Escherichia coli; Fungi such as Aspergillus niger and Candida albicans; and Anaerobes which includes Bacteroides spp, Porphyromonas spp, and Clostridium spp. Crude extracts of all solvents used varied in zones of inhibition. The anaerobes and the gram-positive bacteria were susceptible to all the extracts with minimum inhibitory concentration (MIC) ranging from 32 mg/ml-128 g/ml. The activity against the fungi showed only the oil extract potent for A. niger, while Candida albicans was susceptible to all the extracts with MIC ranging from 256 mg/ml-512 mg/ml. The gram-negatives have MIC ranging from 64 mg/ml-512 mg/ml. Minimum bactericidal concentration (MBC) ranged between 32 mg/ml to 512 mg/ml depending on isolates and extracting solvent. The oil and palm-wine extract of "epa-ijebu" showed greater activity than the other extracts. The killing rate of the schnapps extract on S. aureus and E. coli was 1 and 3.5 hours respectively.

Medicinal plants useful for malaria therapy in Okeigbo, Ondo State, Southwest Nigeria.

There is increasing resistance of malaria parasites to chloroquine, the cheapest and commonly used drug for malaria in Nigeria. Artemisin, a product from medicinal plant indigenous to China, based on active principle of Artemisia annua, has been introduced into the Nigerian market. However not much has been done to project antimalaria properties of indigenous medicinal plants. This study thus, has the main objective of presenting medicinal plants used for malaria therapy in Okeigbo, Ondo State, South west Nigeria. Focus group discussions and interview were held about plants often found useful for malaria therapy in the community. Fifty species (local names) including for example: Morinda lucida (Oruwo), Enantia chlorantha (Awopa), Alstonia boonei (Ahun), Azadirachta indica (Dongoyaro) and Khaya grandifoliola (Oganwo) plants were found to be in use for malaria therapy at Okeigbo, Southwest, Nigeria . The parts of plants used could either be the barks, roots, leaves or whole plants. The recipes also, could be a combination of various species of plants or plant parts. This study highlights potential sources for the development of new antimalarial drugs from indigenous medicinal plants found in Okeigbo, Nigeria.