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Diverse strain types of methicillin-resistant Staphylococcus aureus (MRSA) cause infections in community settings worldwide. To examine heterogeneity of spread within households and to identify common risk factors for household transmission across settings, primary data from studies conducted in New York (USA), Breda (The Netherlands), and Melbourne (Australia) were pooled. Following MRSA infection of the index patient, household members completed questionnaires and provided nasal swabs. Swabs positive for S. aureus were genotyped by spa sequencing. Poisson regression with robust error variance was used to estimate prevalence odds ratios for transmission of the clinical isolate to non-index household members. Great diversity of strain types existed across studies. Despite differences between studies, the index patient being colonized with the clinical isolate at the home visit (P < 0·01) and the percent of household members aged <18 years (P < 0·01) were independently associated with transmission. Targeted decolonization strategies could be used across geographical settings to limit household MRSA transmission.
The transforming growth factor-β (TGF-β) gene family regulates critical processes in animal development, and plays a crucial role in regulating the mammalian immune response. We aimed to identify TGF-β homologues from 2 laboratory model nematodes (Heligmosomoides polygyrus and Nippostrongylus brasiliensis) and 2 major parasites of ruminant livestock (Haemonchus contortus and Teladorsagia circumcincta). Parasite cDNA was used as a template for gene-specific PCR and RACE. Homologues of the TGH-2 subfamily were isolated, and found to differ in length (301, 152, 349 and 305 amino acids respectively), with variably truncated N-terminal pre-proteins. All contained conserved C-terminal active domains (>85% identical over 115 amino acids) containing 9 cysteine residues, as in C. elegans DAF-7, Brugia malayi TGH-2 and mammalian TGF-β. Surprisingly, only the H. contortus homologue retained a conventional signal sequence, absent from shorter proteins of other species. RT-PCR assays of transcription showed that in H. contortus and N. brasiliensis expression was maximal in the infective larval stage, and very low in adult worms. In contrast, in H. polygyrus and T. circumcincta, tgh-2 transcription is higher in adults than infective larvae. The molecular evolution of this gene family in parasitic nematodes has diversified the pre-protein and life-cycle expression patterns of TGF-β homologues while conserving the structure of the active domain.
The sheep scab mite, Psoroptes ovis, induces an intensely pruritic exudative dermatitis which is responsible for restlessness, loss of appetite and weight loss. Within the first 24 h of infection, there is a rapid inflammatory influx of eosinophils and apoptosis of the keratinocytes at the site of infection. The former cell type is capable of a sustained respiratory burst, toxic products of which may directly damage the mite and also contribute to lesion formation. Analysis of a P. ovis expressed sequence tag (EST) database identified a number of antioxidant enzyme-encoding sequences, including peroxiredoxin (thioredoxin peroxidase EC 18.104.22.168), all of which may help the mite endure the potentially toxic skin environment. A full length sequence encoding Po-TPx, a protein of 206 amino acids which showed high homology to a peroxiredoxin from the salivary gland of the tick Ixodes scapularis, was amplified from P. ovis cDNA. Recombinant Po-TPx was expressed in bacteria and antiserum to this protein was used to localize native Po-TPx in mite sections. Peroxiredoxin was localized, amongst other sites, to a subpharyngeal region in mite sections. The recombinant protein was recognized by sera from sheep infested with the mite suggesting that it may be secreted or excreted by the mite and interact with the host immune response.
Negative consumer opinion poses a potential barrier to the application of nutrigenomic intervention. The present study has aimed to determine attitudes toward genetic testing and personalised nutrition among the European public. An omnibus opinion survey of a representative sample aged 14–55+ years (n 5967) took place in France, Italy, Great Britain, Portugal, Poland and Germany during June 2005 as part of the Lipgene project. A majority of respondents (66 %) reported that they would be willing to undergo genetic testing and 27 % to follow a personalised diet. Individuals who indicated a willingness to have a genetic test for the personalising of their diets were more likely to report a history of high blood cholesterol levels, central obesity and/or high levels of stress than those who would have a test only for general interest. Those who indicated that they would not have a genetic test were more likely to be male and less likely to report having central obesity. Individuals with a history of high blood cholesterol were less likely than those who did not to worry if intervention foods contained GM ingredients. Individuals who were aware that they had health problems associated with the metabolic syndrome appeared particularly favourable toward nutrigenomic intervention. These findings are encouraging for the future application of personalised nutrition provided that policies are put in place to address public concern about how genetic information is used and held.
Molecular markers have been used to study genetic diversity within a set of Lablabpurpureus accessions collected from the southern states of India. Amplified fragment length polymorphism (AFLP) molecular marker studies using a total of 78 L. purpureus accessions with nine primer combinations showed there was very little genetic diversity within the L. purpureus accessions from the southern Indian germplasm collection as compared to a set of 15 accessions from other international germplasm collections that included African accessions. The set of 15 were selected from a random amplified length polymorphism (RAPD) marker study and chosen on the basis of widest genetic distance. Further molecular analysis with polymerase chain reaction (PCR) markers from 97 expressed sequence tag (EST) and gene-specific primer pairs, designed from a range of legume sequences, concurred with the AFLP analyses. Both of these approaches provide a wealth of markers for diversity and mapping studies. The 97 sequence-specific primer pairs tested in L. purpureus resulted in 70% amplification success, with 44% of primer pairs amplifying single bands and 10% double bands. Markers generated from these EST and genomic sequences provide useful cross-reference to comparative legume genomics that will potentially have long-term benefit to legume plant breeding.
This paper summarises the progress towards vaccine development against the major blood-feeding nematodes of man and livestock, the hookworms and Haemonchus contortus, respectively. The impact of the diseases and the drivers for vaccine development are summarized as well as the anticipated impact of the host immune response on vaccine design. The performance requirements are discussed and progress towards these objectives using defined larval and adult antigens, many of these being shared between species. Specific examples include the Ancylostoma secreted proteins and homologues in Haemonchus as well as proteases used for digestion of the blood meal. This discussion shows that many of the major vaccine candidates are shared between these blood-feeding species, not only those from the blood-feeding stages but also those expressed by infective L3s in the early stages of infection. Challenges for the future include: exploiting the expanding genome information for antigen discovery, use of different recombinant protein expression systems, formulation with new adjuvants, and novel methods of field testing vaccine efficacy.
The treatment and prevention of parasitism in both humans and livestock continues to rely almost exclusively on the use of antiparasitic drugs – an approach which has limitations, particularly as reinfection, which occurs rapidly in endemic regions, is not prevented. In addition, the widespread appearance of drug-resistant parasites of animals (Kaplan, 2004;) together with emerging evidence of resistance problems in human parasites (Fallon et al. 1995; Ismail et al. 1996; De Clerq et al. 1997; East African Network for Monitoring Antimalarial Treatment, 2003), emphasise the importance of developing alternative methods of control, with anti-parasite vaccines a prime target.
RNA interference (RNAi) on parasitic nematodes has been described as successful and useful for the identification of novel drug and vaccine candidates. In this study we have evaluated this technology on the cattle parasite Ostertagia ostertagi. Eight different genes were targeted in L1 and L3 O. ostertagi larvae, by electroporation and soaking in dsRNA respectively. Down-regulation of target transcript levels was evaluated by semi-quantitative reverse transcriptase (RT) PCR. In L3 larvae, variable decreases in mRNA levels were observed for 5 genes, ranging from a complete knock down (tropomyosin, β-tubulin) to a minor decrease (ATPsynthase, superoxide dismutase, polyprotein allergen). However, repeated experiments indicated that effects were sometimes difficult to reproduce. RNAi for ubiquitin, a transthyretin-like protein and a 17 kDa excretion secretion (ES) protein never resulted in a knock down of the transcript. The mRNA levels of 7 non-target genes showed no difference between larvae soaked in C. elegans control dsRNA versus O. ostertagi tropomyosin dsRNA, supporting that the observed reductions are specific for the target gene. Electroporation of L1 larvae proved to be less effective. Reductions in mRNA levels were only noticed for 2 genes and were not reproducible. In conclusion, the results indicate that the RNAi pathway is probably present in O. ostertagi but that the current RNAi techniques can not be used as a reliable screening method.
Asparaginyl proteinases (or legumains) are a recently identified, novel class of cysteine proteinase which specifically hydrolyse peptide bonds after asparagine residues. Legumains have been implicated in the activation of cysteine proteases, particularly cathepsin B-like proteinases which are thought to help degrade the bloodmeal in blood-feeding helminths such as schistosomes, hookworms and other nematode species. An EST sequence representing a full-length legumain was identified from the Haemonchus contortus datasetNucleotide sequence data described in this paper have been deposited in the EMBL database under the Accession number AM177177.. This encoded a protein with a predicted Mr of 49 kDa, the amino acid sequence of which showed good homology (34–40% identity) to legumains from Schistosoma mansoni, human and rat and contained a legumain-like active site. RT-PCR indicated that the legumain transcript was expressed from the L4 life-cycle stage onwards. The coding sequence was expressed in E. coli and antibodies to the resultant recombinant protein indicated that the enzyme was expressed in the microvillar surface of the intestinal cells. Legumain activity was detected in extracts of the adult parasite but not the host protective Thiol-Sepharose-binding fraction, although it was detectable in the latter by immunoblot. Activity was relatively insensitive to E64, an inhibitor of cysteine proteinases and completely inhibited by the alkylating agent, N-ethylmaleimide, consistent with inhibitor effects on previously characterized legumains.
RNA interference (RNAi) has become an invaluable tool for the functional analysis of genes in a wide variety of organisms including the free-living nematode Caenorhabditis elegans. Recently, attempts have been made to apply this technology to parasitic helminths of animals and plants with variable success. Gene knockdown has been reported for Schistosoma mansoni by soaking or electroporating different life-stages in dsRNA. Similar approaches have been tested on parasitic nematodes which clearly showed that, under certain conditions, it was possible to interfere with gene expression. However, despite these successes, the current utility of this technology in parasite research is questionable. First, problems have arisen with the specificity of RNAi. Treatment of the parasites with dsRNA resulted, in many cases, in non-specific effects. Second, the current RNAi methods have a limited efficiency and effects are sometimes difficult to reproduce. This was especially the case in strongylid parasites where only a small number of genes were susceptible to RNAi-mediated gene knockdown. The future application of RNAi in parasite functional genomics will greatly depend on how we can overcome these difficulties. Optimization of the dsRNA delivery methods and in vitro culture conditions will be the major challenges.
The nature of the proteins which comprise the in vitro excretory/secretory products (ES) of the fourth-stage larva (L4) and adult Teladorsagia circumcincta are largely undefined, despite the fact that this nematode induces profound changes, in part related to parasite ES, in the cellular architecture of the glands lining the abomasal surface of infected sheep and goats. In this study, the protein components of L4 and adult ES were fractionated using 1D gel electrophoresis and the major protein bands, detected by Coomassie blue staining, excised from the gel and subjected to tryptic digest and subsequent mass spectrometric analysis. The resultant peptide mass fingerprints were used to identify 15 L4 and 13 adult ES proteins. Several proteins, such as globin and some metabolic enzymes, were present in both ES. L4 ES alone contained thioredoxin peroxidase, an enzyme that can detoxify free radicals resulting from host inflammatory responses to the parasite, a cysteine proteinase which may aid penetration of the gastric mucosa and 2 different galectins which may influence cell differentiation and morphogenesis. Adult ES contained a nucleoside diphosphate kinase homologue, an enzyme which has been linked to cellular changes and can affect liquid secretion and goblet cell degranulation.
This study evaluated a relatively new molecular technique, serial analysis of gene expression (SAGE), as a tool for quantifying gene expression in the ovine abomasal nematode Haemonchus contortus for which there is relatively limited (~20% gene coverage) sequence information. SAGE technology generates data that are both qualitative and quantitative and, as such, compliments other functional genomics approaches such as EST analysis and micro-array. Prior to embarking on large-scale comparisons, the present study was initiated to establish (i) how well SAGE and EST data taken from the same life-cycle stage would compare, (ii) how easily SAGE tags could be assigned to genes given that the genome sequence is not available and (iii) whether it would be possible to extend the sequences of the SAGE tags to facilitate their identification. Of 2825 tag sequences analysed from adults harvested 28 days post-infection, the identity of the encoding gene could be ascribed to 63% of the tags. The relative abundance of these genes, arbitrarily categorized on the basis of function, was comparable with that of an EST dataset also from adults (n=2317). In addition, tag sequences could be readily extended and thereby identified using a tag-based primer and Reverse Transcription-PCR.
The details of the formation of the first objects, stars and galaxies and their subsequent evolution remain a cosmological unknown. Few observational probes of these processes exist. The Cosmic Infrared Background (CIB) originates from this era and measurements of its anisotropy can provide information to test models of both galaxy evolution and the growth of primordial structure. Such measurements should provide a sensitive probe of the large-scale variation in protogalaxy density at redshifts, z ~ 0.5-3, while optical galaxy surveys provide complementary information at z < 0.5 and Lyman alpha absorption forest studies and Cosmic Microwave Background measurements add information at higher redshifts.
A recent study carried out by Houdijk et al (2005), used a rodent model to assess whether a reduction in protein scarcity during lactation resulted in a reduced degree of parasitism. Feeding high protein foods resulted in a reduced worm burden, but was confounded with increased food intake per se. Therefore, effects observed on parasitism may not necessarily have been associated with an increased protein supply, but with changes in the gut environment due to the increased food intake. Before this model can be used to assess the underlying immune responses, further work is needed to verify that the effects observed are indeed related to changes in nutrient supply. This experiment aimed to provide further evidence on the nutritional control of parasitism during lactation by manipulating nutrient demand. It was expected that the latter would not be associated with changes in food intake per se and results could therefore be used to exclude the influence of non-immunological changes in the gut environment as a contributing factor of reduced parasitism.
Members of the genus Trichostrongylus, such as T. vitrinus, being endemic in Northern Europe, are among the principal causative nematodes which contribute to parasitic gastro-enteritis in sheep world-wide, inhabiting the proximal small intestine and causing damage to the mucosa. This results in impaired nutrient absorption as well as a pronounced inflammatory response with cellular infiltration of the mucosa, including a pronounced mast cell response. These mast cells release serine proteinases that enhance the passage of effector cells and macromolecules across epithelial boundaries and into direct contact with the invading parasite. The adult and larval stages of T. vitrinus release a number of serine proteinases in vitro that may contribute to tissue invasion and nutrient acquisition in vivo. This study describes the molecular cloning and characterization of a serine proteinase inhibitor (serpin)Sequence reported here is available with GenBank Accession number Y12233. that is present in extracts of all the parasitic stages, becoming more abundant as the life-cycle progresses. The serpin is present in the in vitro excretory/secretory products (ES) of 4th-stage larval and adult parasites, being more abundant in the former. The serpin was expressed in E. coli and the recombinant protein was a potent inhibitor of several host serine proteinases including mast cell proteinases. The serpin may regulate the activity of the parasite serine proteinases or it may modulate the host immune response to the parasite by inhibiting the activity of serine proteinases released from host inflammatory cells.
Psoroptes ovis, the causative agent of sheep scab, is an important ectoparasitic mite infecting sheep, goats and cattle. Infection is characterized by an extensive dermatitis, scab formation and intense itching. Initial focal lesions spread outwards, coalesce and may extend over the whole body. The host response to infestation has all the characteristics of an immediate-type hypersensitivity reaction but the mite antigens and allergens which initiate this response are almost completely undefined. Here, 507 randomly selected cDNAs derived from a mixed population of P. ovis were sequenced and the resultant nucleotide sequences subjected to Cluster analysis and Blast searches. This analysis yielded 280 clusters of which 49 had >1 sequence with 24 showing significant Blast X homology to another protein in the databases. There were 231 sequences which appeared on one occasion and 109 of these showed significant Blast X homology to other sequences in the databases. This analysis identified homologues of 9 different types of allergens which have been characterized in other allergic conditions such as responses to house dust mites. It also identified a number of cysteine proteases which may contribute to lesion development as well as several free-radical scavenging enzymes which may protect the mite from host immune effector responses.