Chromosomal mapping of host susceptibility loci to Angiostrongylus costaricensis nematode infection in mice

Angiostrongylus costaricensis is a nematode found mainly as a rodent parasite. Laboratory mice were experimentally infected with this parasite. It is known that there is great variability in mortality among inbred mouse strains after infection with this nematode. The survival rate at 5 weeks after infection of A/J mice was 90.5%, whereas that of SM/J mice was only 33.3%, with severe anemia and decreased body weight about 3 weeks after infection. To identify host susceptibility genes for infection with this nematode, we undertook chromosomal mapping by a whole-genome scanning approach in (A/JxSM/J)F2 mice. We mapped a host susceptibility locus (here designated Acsns, for Angiostrongylus costaricensis nematode susceptibility locus) to the telomeric portion of Chromosome 19 (peak LOD=4.35). We also identified two loci on Chr 13 and Chr 17 that have epistatic effects on host survival. This is the first report on host susceptibility loci for helminth infection mapped by whole-genome scanning.     

Characterization of Angiostrongylus cantonensis excretory-secretory proteins as potential diagnostic targets

Angiostrongyliasis results from infections with intra-arterial nematodes that accidentally infect humans. Specifically, infections with Angiostrongylus cantonensis cause eosinophilic meningitis and Angiostrongylus costaricensis infections result in eosinophilic enteritis. Immunological tests are the primary means of diagnosing infections with either pathogen since these parasites are usually not recoverable in fecal or cerebrospinal fluid. However, well-defined, purified antigens are not currently available in sufficient quantities from either pathogen for use in routine immunodiagnostic assays. Since A. costaricensis and A. cantonensis share common antigens, sera from infected persons will recognize antigens from either species. In addition to their potential use in angiostrongyliasis diagnosis, characterization of these proteins that establish the host-parasite interphase would improve our understanding of the biology of these parasites. The main objective of the present work was to characterize A. cantonensis excretory-secretory (ES) products by analyzing ES preparations by two-dimensional gel electrophoresis coupled with immunoblotting using pools of positive sera (PS) and sera from healthy individuals (SC). Protein spots recognized by PS were excised and analyzed by electrospray ionization (ESI) mass spectrometry. MASCOT analysis of mass spectrometry data identified 17 proteins: aldolase; CBR-PYP-1 protein; beta-amylase; heat shock protein 70; proteosome subunit beta type-1; actin A3; peroxiredoxin; serine carboxypeptidase; protein disulfide isomerase 1; fructose-bisphosphate aldolase 2; aspartyl protease inhibitor; lectin-5; hypothetical protein F01F1.12; cathepsin B-like cysteine proteinase 1; hemoglobinase-type cysteine proteinase; putative ferritin protein 2; and a hypothetical protein. Molecular cloning of these respective targets will next be carried out to develop a panel of Angiostrongylus antigens that can be used for diagnostic purposes and to further study host-Angiostrongylus interactions.     

Use of heterologous antigens for the immunodiagnosis of abdominal angiostrongyliasis by an enzyme-linked immunosorbent assay

Angiostrongylus costaricensis has a broad geographic distribution spanning from North to South America and the infections of vertebrates with this nematode can result in abdominal complications. Human infections are diagnosed by histological or serological methods because the isolation of larvae from feces is not feasible, as most parasites become trapped in intestinal tissues due to intense eosinophilic inflammation. Because A. costaricensis is difficult to maintain in the laboratory, an immunodiagnostic IgG enzyme-linked immunosorbent assay (ELISA) using antigens from the congeneric Angiostrongylus cantonensis species was evaluated against a panel of serum samples from patients who were histologically diagnosed with A. costaricensis infections. Sera from uninfected individuals and individuals infected with other parasites were used as controls. The sensitivity and specificity of the assay were estimated at 88.4% and 78.7%, respectively. Because the use of purified or cloned antigens has not been established as a reliable diagnostic tool, the use of heterologous antigens may provide a viable alternative for the development of an ELISA-based immunodetection system for the diagnosis of abdominal angiostrongyliasis.     

Isolation of Angiostrongylus costaricensis first-stage larvae from rodent feces on a Percoll gradient

Several density gradients were tested for the isolation of parasitic nematode, Angiostrongylus costaricensis, first-stage larvae from rodent feces. With a 45/72% Percoll gradient, 83-99% (89.56+/-6.57%) of the larvae were recovered in a clean preparation.     

Alteration in the endogenous intestinal flora of Swiss Webster mice by experimental Angiostrongylus costaricensis infection

The association between worm infections and bacterial diseases has only recently been emphasized. This study examined the effect of experimental Angiostrongylus costaricensis infection on endogenous intestinal flora of Swiss Webster mice. Eight mice aging six weeks were selected for this experiment. Four were infected with A. costaricensis and the other four were used as controls. Twenty eight days after the worm infection, all mice in both groups were sacrificed and samples of the contents of the ileum and colon were obtained and cultured for aerobic and anaerobic bacteria. In the mice infected with A. costaricensis there was a significant increase in the number of bacteria of the endogenous intestinal flora, accompanied by a decrease in the number of Peptostreptococcus spp. This alteration in the intestinal flora of mice infected by the nematode may help to understand some bacterial infections described in humans.     

Public health importance of Angiostrongylus cantonensis and its relatives

Species of Angiostrongylus are fairly common nematode parasites of a variety of small mammals, but some species - particularly A. cantonensis and A. costaricensis - are increasingly seen as human infections. The worms make use of a range of aquatic, amphibious or terrestrial gastropod molluscs as intermediate hosts, which become infected by ingestion of eggs shed in the faeces of infected mammals. Porotenic hosts, including crustaceans, amphibians and reptiles, are known for most Angiostrongylus species, and can also be a source of the infective third-stage larvae that initiate mammalian infections.     

Angiostrongylus costaricensis egg antigen for the immunodiagnosis of abdominal angiostrongyliasis

Angiostrongylus costaricensis is the aetiological agent of human abdominal angiostrongyliasis, a parasitic disease reported from the United States to Argentina, with a widespread occurrence of the nematode throughout Central and South America. This study assesses the performance of A. costaricensis eggs as antigen in an enzyme-linked immunosorbent assay (ELISA), for the determination of parasite-specific IgG1 antibodies. The specificity and the sensitivity of the method were 87% and 90.5%, respectively. Through this test it was possible to demonstrate a sharp and early decline in IgG1 antibody in serum samples taken from patients with histopathological diagnosis of abdominal angiostrongyliasis at different time points after surgical treatment. The present work demonstrated the usefulness of the egg antigen in the development of a specific diagnostic test for abdominal angiostrongylosis.     

Phyllocaulis variegatus--an intermediate host of Angiostrongylus costaricensis in south Brazil

Molluscs collected in five localities in the State of Rio Grande do Sul (Brazil) were digested and examined. The infected slugs were identified as Phyllocaulis variegatus and the larvae found were inoculated per os into mice. After 50 days, worms with the caracteristics of Angiostrongylus costaricensis were recovered from the mesenteric arterial system. The results establish the role of P. variegatus as intermediate host of A. costaricensis in south Brazil, where many cases of abdominal angiostrongyliasis have been diagnosed.     

Spontaneous occurrence of Angiostrongylus costaricensis in marmosets (Saguinus mystax)

Two marmosets imported from Iquitos, Peru, were found to be infected with Angiostrongylus costaricensis. Both animals had large solitary granulomas involving the wall and adjacent mesentery of the small intestine. Histopathologic examination showed the adult nematodes in the lumina of the mesenteric arteries that coursed through these granulomas. The inflammatory reaction was associated with numerous degenerating eggs and larvae. This is the first report of this parasite in nonhuman primates and extends its geographic range to Peru. In addition, in one animal, Dipetalonema sp were seen free in the abdominal cavity, and pleroceroid larvae (spargana) were in the loose connective tissue of the left axilla. This animal also had microgranulomas associated with eggs and larvae of Angiostrongylus in the kidney, liver, lung, and heart.     

Proteomic signature of human embryonic stem cells

Human embryonic stem cells (hESC) represent a population of undifferentiated pluripotent cells with both self-renewal and multilineage differentiation characteristics. Proteomics provides a powerful approach for studying the characteristics of hESC and discovering molecular markers. We have analyzed proteome profiles of three hESC lines using 2-DE and MALDI TOF-TOF. Out of 844 spots analyzed with MALDI TOF-TOF, 685 proteins were identified of which 60 proteins were classified as the most abundant proteins on 2-D gels. A large number of proteins particularly high abundant ones were identified as chaperones, heat shock proteins, ubiquitin/proteasome, and oxidative stress responsive proteins underscoring the ability of these cells to resist oxidative stress and increase the life span. Several proteins involved in cell proliferation and differentiation were also among the highly expressed proteins. Although overall expression pattern of three hESC were similar, 54 spots changed quantitatively and 14 spots changed qualitatively among the hESC cell lines. Most of these proteins were identified as proteins involved in cell growth, metabolism and signal transduction, which may affect the self-renewal and pluripotency. To our knowledge, this study represents the first proteomic dataset for hESC and provides a better insight into the biology of hESC. Proteome maps of hESC are accessible at http://www.RoyanProteomics.ir.     

Differential proteomics analysis of female and male adults of Angiostrongylus cantonensis

In this study, we identified the differentially expressed proteins of female and male adults of Angiostrongylus cantonensis through differential proteomics. We extracted and purified total proteins from male and female adults, separated proteins by two-dimensional difference gel electrophoresis (2D-DIGE) in pH 4-7, analyzed the gel images by DeCyder 7.0 software, and sacrificed the infected rats to count the number of male and female adults. It was found 28 protein spots that were differentially expressed; seven protein spots were then identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Five proteins were up-regulated and two proteins down-regulated in male adults compared with female adults. Three of the five up-regulated proteins with known functions ascribed to them were identified as galectin-1, proteasome alpha subunit and peroxiredoxin. The two down-regulated proteins were identified as indoleamine dioxygenase like-myoglobin and galectin. Furthermore, the female was significantly greater than male adults (P<0.01) in the rats. The findings demonstrate the differences in protein expression profiles and the ability to survive in the final host between female and male adults of A. cantonensis, and may provide a theoretical basis to study their developmental biology further.     

Proteomic Analysis of Osteoblasts Exposed to Fluoride In Vitro

Proteomical analysis is defined as the characterization of the entire set of protein encoded a genome. Two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) are main techniques used in proteomic analysis to achieve information about protein expression profiles. Knowledge about the mechanism of skeletal fluorosis can be gained by recognizing changes in protein expression. To better understand the skeletal fluorosis process, osteoblasts isolated from calvarial of neonatal mouse were cultured and treated with 2 ppm fluoride for 72 h, and proteins of the osteoblast were profiled by 2-DE. With the analysis of Image-Master 2D analysis software, we detected a total number of 493 matching spots on 2-DE images. Among them, 28 protein spots showed twofold significant alteration (P < 0.05) in fluoride-exposed groups. Moreover, 12 proteins were identified by MALDI-TOF MS. These identified proteins in fluoride-exposed group were associated with cell proliferation, metabolism, and oxidative folding. Thus, our study provides useful information on fluoride-related changes of proteome and shows that proteomical analysis is a powerful methodology for the better understanding of skeletal fluorosis.     

Proteomics of extreme freezing tolerance in Siberian spruce (Picea obovata)

Differential expression of proteins in needles of the extreme freeze tolerant conifer Picea obovata during September, October and November was analyzed using DIGE technology and multivariate analysis. More than 1200 spots were detected, and the abundance of 252 of these spots was significantly altered during the course of acclimation. The 252 spots were clustered into five distinct expression profiles. Among the protein spots showing differential expression, 43 were identified by MALDI-TOF/TOF and twelve of them matched proteins associated with various biotic and abiotic stress responses in other plants. Dehydrins, Hsp70s, AAA⁺ ATPases, lipocalin, cyclophilins, glycine-rich protein (GNP) and several reactive oxygen intermediate scavenging proteins showed increased accumulation levels from September to November. The expression profiles and putative role of the identified proteins during acclimation and freezing tolerance are discussed.     

Molecular differentiation of Angiostrongylus costaricensis, A. cantonensis, and A. vasorum by polymerase chain reaction-restriction fragment length polymorphism

Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2) and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.     

Angiostrongylus costaricensis and experimental infection of Sarasinula marginata II: elimination routes

Angiostrongylus costaricensis intermediate hosts are terrestrial mollusks mostly belonging to the Veronicellidae family. In the present investigation we focused on the mechanisms of larval expulsion from Sarasinula marginata infected with A. costaricensis. Twenty-five mollusks were individually infected with 5000 L1 and sacrificed at 30 min and 1, 2, 4, 6, and 8 h post-infection and at days 1, 2, 4, 5, 6, 8, 10, 11, 12, 14, 15, 16, 20, 21, 22, 25, 26, 28, and 30 post-infection; the mollusks were then fixed and stained. Diverse organs involved throughout the course of the migratory routes of larvae from oral penetration on were specified and the mechanisms of larval access to the fibromuscular layer through the kidney, rectum, and vascular system were defined. The elimination of L3, derived from oral and/or cutaneous infections, appears to depend on granulomas located close to the excretory ducts of mucous cells.     

滤减UV-B辐射对烤烟蛋白质组变化的影响

为研究不同UV-B辐射强度对烤烟生理代谢及调控途径的影响,应用蛋白质双向电泳联用质谱技术,以云南普遍种植的烤烟K326为试验材料,通过覆盖不同透明薄膜滤减UV-B辐射的方式,对75.8%(聚乙烯膜,处理1)和37.5%(麦拉膜,处理2)UV-B辐射透过率处理下K326的蛋白质组和相关生理性状进行了比较。结果表明:在蛋白质组中有10个蛋白在这两类处理下蛋白差异表达显著;与处理1相比,在处理2的K326叶片中有5个蛋白上调表达,5个蛋白下调表达;通过质谱分析共鉴定出8种功能明确的蛋白质,其中差异表达的10个蛋白中有3个与氧化还原相关,3个与光合作用相关,1个是参与能量代谢的激酶蛋白,1个是RNA结合蛋白,另外还有2个未知功能的蛋白待探明,在蛋白质组水平对不同UV-B辐射强度与烤烟生长发育的关系进行了初步研究;而在K326的生理成熟期、过渡期及工艺成熟期,处理2的净光合速率(Pn)均高于处理1,这与所鉴定出的3个与光合作用有光的蛋白在处理2中上调表达趋势一致;相比之下,处理1的K326发育进程较快,茎粗等形态指标及比叶重均比处理2高。     

Developmental control of stress stimulons in Streptomyces coelicolor revealed by statistical analyses of global gene expression patterns

Stress-induced regulatory networks coordinated with a procaryotic developmental program were revealed by two-dimensional gel analyses of global gene expression. Four developmental stages were identified by their distinctive protein synthesis patterns using principal component analysis. Statistical analyses focused on five stress stimulons (induced by heat, cold, salt, ethanol, or antibiotic shock) and their synthesis during development. Unlike other bacteria, for which various stresses induce expression of similar sets of protein spots, in Streptomyces coelicolor heat, salt, and ethanol stimulons were composed of independent sets of proteins. This suggested independent control by different physiological stress signals and their corresponding regulatory systems. These stress proteins were also under developmental control. Cluster analysis of stress protein synthesis profiles identified 10 different developmental patterns or "synexpression groups." Proteins induced by cold, heat, or salt shock were enriched in three developmental synexpression groups. In addition, certain proteins belonging to the heat and salt shock stimulons were coregulated during development. Thus, stress regulatory systems controlling these stimulons were implicated as integral parts of the developmental program. This correlation suggested that thermal shock and salt shock stress response regulatory systems either allow the cell to adapt to stresses associated with development or directly control the developmental program.     

First record of molluscs naturally infected with Angiostrongylus cantonensis (Chen, 1935) (Nematoda: Metastrongylidae) in Brazil

Seeking the identification of Angiostrongylus cantonensis as a potential etiological agent of three clinical cases of eosinophilic meningitis, mollusc specimens were collected in the state of Espírito Santo, Brazil. The snails were identified as Sarasinula marginata (45 specimens), Subulina octona (157), Achatina fulica (45) and Bradybaena similaris (23). Larvae obtained were submitted to polymerase chain reaction and restriction fragment length polymorphism diagnosis. Their genetic profile were corresponded to A. cantonensis. Rattus norvegicus experimentally infected with third-stage larvae, developed menigoencephalitis, and parasites became sexually mature in the lungs. Additionally, larvae obtained from A. fulica snails, from S?o Vicente, state of S?o Paulo, also showed genetic profiles of this nematode. This is the first record of Brazilian molluscs infected with this nematode species.