Ethanol increases HSP70 concentrations in honeybee (Apis mellifera L.) brain tissue

Previous research on the honeybee ethanol model established how acute ethanol exposure altered function at different levels of organization: behavior and learning, ecology, and physiology. The purpose of this study was to evaluate whether ethanol doses that affect honeybee behavior also induce a significant stress response, measured by heat shock protein 70 (HSP70) concentrations, in honeybee brain tissues. Experiment 1 examined how pretreatment handling influenced brain HSP70 concentrations in three pretreatment groups of bees; immediately after being collected, after being harnessed and fed, and after 22-24 h in a harness. HSP70 concentrations did not differ among pretreatment groups within replicates, although we observed significantly different HSP70 concentrations between the two replicates. Experiment 2 investigated the relationship between ethanol dose and brain HSP70 concentrations. Bees were placed in seven experimental groups, the three pretreatment groups as in Experiment 1 and four ethanol-fed groups. Bees in ethanol treatments were fed 1.5 M sucrose (control) and 1.5 M sucrose-ethanol solutions containing 2.5, 5, and 10% ethanol, allowed to sit for 4 h, and dissected brains were assayed for HSP70. We observed ethanol-induced increases in honeybee brain HSP70 concentrations from the control group through the 5% ethanol group. Only bees in the 5% ethanol group had HSP70 concentrations significantly higher than the control group. The inverted U-shaped ethanol dose-HSP70 concentration response curve indicated that ingestion of 2.5% ethanol and 5% ethanol stimulated the stress response, whereas ingestion of 10% ethanol inhibited the stress response. Doses that show maximum HSP70 concentration (5% ethanol) or HSP70 inhibition (10% ethanol) correspond to those (≥5% ethanol) that also impaired honeybees in previous studies. We conclude that acute ethanol intoxication by solutions containing ≥5% ethanol causes significant ethanol-induced stress in brain tissue that impairs honeybee behavior and associative learning.     

Characterization of honey bee sensitivity to ethanol vapor and its correlation with aggression.

Several candidate genes identified from quantitative trait loci (QTL) for defensive behavior in honey bees (Apis mellifera L.) are homologous to genes known to influence ethanol sensitivity in other organisms. To investigate this possible link between aggression/defense and ethanol sensitivity, assays were developed to evaluate ethanol vapor responses in worker bees from a low-defensive (gentle) colony and a high-defensive colony. Defensive workers exhibited characteristic signs of ethanol-induced sedation significantly faster than gentle workers upon exposure to ethanol vapor. Backcross workers displayed ethanol sensitivity intermediate to the parental defensive and gentle lines, suggesting a genetic basis for the trait. Workers were screened with sequence-tagged site markers linked to three defensive-behavior QTL and their genotypes were tested for associations with ethanol sensitivity. There were no significant associations, indicating that the defensive QTL were not having a pleiotropic effect on ethanol sensitivity. It is possible that gentle-source alleles at these QTL are dominant with respect to sensitivity, one or more of these QTL were not segregating in the backcross family, or unidentified QTL are influencing alcohol sensitivity.     

Nitrite toxicity to crayfish, Astacus leptodactylus, the effects of sublethal nitrite exposure on hemolymph nitrite, total hemocyte counts, and hemolymph glucose

The 48-h acute toxicity range of nitrite to narrow-clawed crayfish, Astacus leptodactylus was within 22 and 70 mg L(-1) (mean 29.43 mg L(-1)). Environmental chloride (100 mg L(-1) chloride) increased the 48-h toxicity of nitrite to a range of 31 and 80 mg L(-1) (mean 49.20 mg L(-1)). Hemolymph nitrite, total hemocyte counts (THCs), and hemolymph glucose were examined in A. leptodactylus exposed to different sublethal nitrite concentrations. The same parameters were also determined for A. leptodactylus exposed to different sublethal nitrite concentrations with additional environmental chloride. Additionally, hemolymph nitrite and THCs were analyzed for crayfish exposed to nitrite-free water after 24 h following a 48-h exposure to nitrite. In the nitrite-exposed tests, hemolymph nitrite increased directly with water nitrite; however, after recovery, nitrite in hemolymph decreased. In the nitrite plus chloride-exposed tests, the accumulation of nitrite in hemolymph was relatively low compared to the nitrite-exposed tests. Thus, hemolymph to environment ratios of nitrite in the nitrite-exposed tests were higher than those of nitrite plus chloride-exposed tests. THCs decreased following nitrite exposure and, in general, increased after recovery. In the nitrite with chloride exposed and recovery from nitrite tests, THCs increased. Hemolymph glucose levels elevated following nitrite exposure, independent of water nitrite concentrations. However, with environmental chloride nitrite exposure did not cause elevation of hemolymph glucose. Hemolymph nitrite accumulation was found to be closely related to the decrease in THCs and increase in hemolymph glucose.     

A comparison of Val81Met and other polymorphisms of alcohol metabolising genes in patients and controls in Northern Spain

The aim of this paper is to study polymorphism in the TH, ADH1B, ADH1C, ALDH2 and CYP2E1 genes so as to ascertain whether it is associated with excessive consumption of alcohol. The SNPs rs6356 of TH, rs1229984, rs2066702 of ADH1B; rs698, rs1693482 of ADH1C; rs671 of ALDH2; rs72559710, rs55897648, rs6413419, rs3813867, rs2031920, rs6413432 of CYP2E1 were studied in a sample of 172 high-level patients and 150 fully non-drinkers controls. Genotyping was performed using Rt-PCR with Taqman probes. SNPs located at ALDH2 and CYP2E1 showed no heterozygosity. Frequency distribution showed significant differences between the two groups studied for loci TH and ADH1B. The genotype Val/Val of TH locus increased in risk 1.988 times (95% CI: 1.006–3.930) that the subjects carrying the genotype Met/Met; and the genotype ADH1B*1/*1 of ADH1B locus increased in risk 3.811 times (CI: 1.660–8.749) that the subjects carrying the genotype ADH1B*1/*2. Alleles Val and ADH1B*1 may therefore increase the risk of the onset and development of this illness.     

Early treatment failure in severe malaria resulting from abnormally low plasma quinine concentrations

A patient admitted with severe Plasmodium falciparum malaria in western Thailand had an early treatment failure with quinine, despite full dosing. Plasma quinine concentrations were subtherapeutic. Abnormal quinine pharmacokinetics may explain sporadic reports of quinine treatment failures in severe malaria.     

The isolation of Leishmania tropica and L. aethiopica from Phlebotomus (Paraphlebotomus) species (Diptera: Psychodidae) in the Awash Valley, northeastern Ethiopia

In a survey of Leishmania infections in phlebotomine sandflies in a highly suspected focus of leishmaniasis in the Awash Valley (northeastern Ethiopia) between January 1994 and August 1997, a total of 3307 females of 11 Phlebotomus species (P. orientalis, P. fantalensis, P. saevus, P. sergenti, P. gemetchi, P. alexandri, P. bergeroti, P. duboscqi, P. arabicus, P. martini, and P. rodhaini) were dissected. Promastigotes were detected in 17 females of three species (11 P. saevus, 4 P. sergenti and 2 P. arabicus). Of these, only two P. saevus (one from Upper Awash and one from Middle Awash) and three P. sergenti (from Upper Awash) positives were successfully isolated in culture and were typed by isoenzyme analysis. Four isolates (two each from P. saevus and P. sergenti) were identified as new zymodemes (Z) of L. tropica and one isolate from P. sergenti was typed as a new zymodeme of L. aethiopica. This is the first finding of natural infections of P. saevus and P. arabicus and the first evidence for the former to be a vector of L. tropica. This is also the first time P. sergenti has been implicated in L. tropica transmission in Ethiopia; the isolation of L. aethiopica from a Paraphlebotomus species (P. sergenti) is also a new record. The possible presence of human cutaneous leishmaniasis (L. tropica and L. aethiopica), and wild reservoir host(s) of the parasites, especially rock hyrax (Procavia capensis) in the Upper and Middle Awash Valley remain to be determined.     

Antibody levels against B. pertussis in neonates measured in dried blood spots

Aim

We designed this study to investigate if immunoglobuline G-Pertussis toxin (IgG-PT) against Bordetella pertussis in umbilical cord blood can reliably be determined in dried blood spots on filter paper (Guthrie) cards.

Patients and methods

We prospectively included 129 mothers and their newborns born in a general hospital in the Netherlands. The relation between IgG-PT against B. pertussis from the umbilical cord measured in dried blood spots (Guthrie card) and in serum samples was studied by means of a Bland–Altman graph, using regression analysis to evaluate the level of agreement of both measurement methods.

Results

IgG-PT in Guthrie cards show a high coefficient of correlation with IgG-PT in serum samples from the umbilical cord when calibrated against blood spot calibrators (p < 0.05).

Conclusion

Maternal IgG-PT against B. pertussis measured in cord blood applied to Guthrie cards and calibrated against blood spot calibrators show good agreement with measurement of IgG-PT in cord serum. This offers new perspectives for future studies concerning B. pertussis antibodies.     

Heavy metals in flamingos (Phoenicopterus ruber) from Italian wetlands: the problem of ingestion of lead shot

Waterbirds are exposed to many contaminants, including lead from ingestion of shot and fishing sinkers. Lead poisoning had never been reported in flamingos wintering in Italian wetlands. Our investigation stems from a case of four flamingos found dead in Tuscany in 2002 with numerous lead shot in their gizzards. We therefore considered other specimens found dead in different Italian wetlands. Many lead shot found in gizzards and lead tissue concentrations confirmed the hypothesis of lead poisoning in two of the seven specimens analysed: concentrations in liver, kidney, and bone were 361.3, 265.09, and 43.31 μg/g d.w., respectively. Lead organotropism was typical of acute poisoning. Cadmium and mercury were also determined, and found to be in line with what little data are available on this species in the literature.Although Italy recently endorsed the African-Eurasian Waterbird Agreement (AEWA) prohibiting use of lead shot for hunting in wetlands, our results reveal a first case of lead shot poisoning in flamingos wintering in Italian wetlands. This evidence sounds a further warning of the problem of spent lead shot in countries where hunting in wetlands is not strictly regulated.     

Lipopolysaccharide from Burkholderia thailandensis E264 provides protection in a murine model of melioidosis

Burkholderia thailandensis is a less virulent close relative of Burkholderia pseudomallei, a CDC category B biothreat agent. We have previously shown that lipopolysaccharide (LPS) extracted from B. pseudomallei can provide protection against a lethal challenge of B. pseudomallei in a mouse model of melioidosis. Sugar analysis on LPS from B. thailandensis strain E264 confirmed that this polysaccharide has a similar structure to LPS from B. pseudomallei. Mice were immunised with LPS from B. thailandensis or B. pseudomallei and challenged with a lethal dose of B. pseudomallei strain K96243. Similar protection levels were observed when either LPS was used as the immunogen. This data suggests that B. thailandensis LPS has the potential to be used as part of a subunit based vaccine against pathogenic B. pseudomallei.     

Polymorphisms in cg2 and pfcrt genes and resistance to chloroquine and other antimalarials in vitro in Plasmodium falciparum isolates from Colombia

Polymorphisms in Plasmodium falciparum cg2 and pfcrt genes and their association with chloroquine resistance in vitro in Colombian parasites were evaluated in this study. Association of chloroquine resistance with resistance to other antimalarial drugs in vitro was also examined. Polymerase chain reactions (PCR) for kappa and omega cg2 regions and nested PCR and digestion with ApoI enzyme for K-76T pfcrt point mutation defined corresponding polymorphisms in 83 samples collected between 1995 and 1999. The isotopic microtest was used to evaluate sensitivity in vitro in a subgroup of 18 isolates. The predominant cg2 pattern observed was 13K/14omega repeats (46/83 [55.4%]) and all samples presented the K-76T mutant allele. Seventy-eight percent of samples were resistant to chloroquine in vitro, 35.3% to amodiaquine, 16.7% to mefloquine, and 5.6% to quinine. Significant correlations (P < 0.05) were observed between the IC50s of chloroquine and arteether, and among IC50s of arteether, mefloquine, and quinine. These results suggest the development of multiple and cross-resistance of Colombian P. falciparum isolates to second- and third-line antimalarials and new alternative drugs.     

Chronic alcohol ingestion alters claudin expression in the alveolar epithelium of rats.

Previously we determined that chronic alcohol ingestion (6 weeks) in rats increases lung epithelial permeability in vivo approximately 5-6-fold and promotes flooding of the alveolar airspaces with proteinaceous fluid in response to stresses such as sepsis. In parallel, alveolar epithelial cells isolated from alcohol-fed rats fail to form tight monolayers in vitro, even when cultured for up to 8 days in the absence of alcohol. However, the molecular mechanisms underlying alcohol-induced permeability are unknown. Claudins are key components of tight junctions that restrict the paracellular movement of water, proteins, and solutes across cellular barriers including the alveolar epithelium. In this study, we examined the expression of multiple members of the claudin protein family in the lungs of alcohol-fed versus control-fed rats (Lieber-DeCarli liquid diet with either 36% of calories as alcohol or an isocaloric substitution with maltin-dextrin for 6 weeks). We determined that chronic alcohol ingestion affected the expression of multiple claudins; most striking were decreases in claudin-1 and claudin-7, and an increase in claudin-5, in the whole lung and in alveolar epithelial monolayers derived from alcohol-fed rats. In parallel, immunocytochemistry of alveolar epithelial monolayers from alcohol-fed rats revealed abnormal intracellular accumulation of claudin-7 protein and relatively decreased localization to cell membranes. Claudin-1 and claudin-7 are relatively specific to alveolar epithelial type I pneumocytes that form the vast majority of the alveolar epithelial barrier in vivo, and increases in claudin-5 have been associated with increased epithelial permeability in other systems. Therefore, these findings suggest that changes in claudin expression in the alveolar epithelium produce a "leakier" phenotype that renders the alcoholic lung susceptible to alveolar flooding during acute inflammatory stresses.     

Evaluation of a species-specific PCR assay for the Anopheles funestus group from eleven African countries and Madagascar

A newly published cocktail polymerase chain reaction (PCR) assay can identify five members of the Anopheles funestus group: An. funestus, An. vaneedeni, An. parensis, An. leesoni and An. rivulorum. The assay was evaluated on specimens from 11 African countries: Angola, Cote d'Ivoire, Ethiopia, Kenya, Malawi, Mozambique, Namibia, South Africa, Tanzania, Uganda and Zambia; and the island of Madagascar. The polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) and the internal transcribed spacer PCR (ITS2-PCR) assays were used as a priori identification methods on 900 specimens. Of these, 96.4% were correctly identified using the new cocktail PCR method. The remaining 3.5% (32) from Malawi failed to amplify. The failure to identify these samples was not significantly different from the samples from the other countries (Chi-square; P > 0.05). The results suggest that the new species-specific PCR assay is an efficient and effective means of identifying 5 members of the An. funestus group in a single reaction. It is also less time-consuming than the molecular methods previously used on the group.     

Rats as indicators of the presence and dispersal of six zoonotic microbial agents in Cyprus, an island ecosystem: a seroepidemiological study

A total of 622 rats (402 Rattus norvegicus and 220 R. rattus frugivorus) were collected in 51 different areas in Cyprus during 2000-2003 and used as indicators of the presence and dispersal of six zoonotic microbial agents. IgG antibodies against Rickettsia typhi (241/496, 48.6%), R. conorii (209/500, 41.8%), Toxoplasma sp. (138/494, 27.9%), Coxiella burnetti (63/494, 12.8%), Bartonella henselae (52/494, 10.5%) and Leishmania infantum (36/494, 7.3%) were detected by indirect immunofluorescence test. There was variation in the association between the seropositivity of the six microbial agents and other factors. Rat species affected R. typhi and R. conorii seropositivity, the prefecture where the rats were caught affected R. typhi, C. burnetii, B. henselae, T. gondii and L. infantum, the sampling season impacted on R. typhi, R. conorii, T. gondii and L. infantum, and the flea species affected R. typhi, R. conorii and B. henselae. These results were analysed using geographical information system (GIS) technology and the seropositivity in rats against the pathogens tested appeared to follow the occurrence of these pathogens in humans. This suggests that rats could be used as disease sentinels and, together with GIS technology, they could be a useful tool for the identification of endemic foci and high-risk areas for each pathogen.     

In vitro assessment of halobacterial gas vesicles as a Chlamydia vaccine display and delivery system

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted disease worldwide and while antibiotic treatment is effective in eliminating the pathogen, up to 70% of all infections are asymptomatic. Despite sustained efforts over the past 2 decades, an effective chlamydial vaccine remains elusive, due in large part to the lack of an effective delivery system. We explored the use of gas vesicles derived from Halobacterium salinarium as a potential display and delivery vehicle for chlamydial antigens of vaccine interest. Various size gene fragments coding for the major outer membrane protein (MOMP), outer membrane complex B (OmcB) and polymorphic outer membrane protein D (PompD) were integrated into and expressed as part of the gas vesicle protein C (gvpC) on the surface of these stable structures. The presence of the recombinant proteins was confirmed by Western blots probed using anti-gvpC and anti-Chlamydia antibodies as well as sera from Chlamydia-positive patients. Tissue culture evaluation revealed stability and a time-dependent degradation of recombinant gas vesicles (r-Gv) in human and animal cell lines. In vitro assessment using human foreskin fibroblasts (HFF) confirmed Toll-like receptor (TLR) 4 and 5 engagement by wild type and r-Gv, leading to MyD88 activation, TNF-α, IL-6 and IL-12 production. The data suggest that r-GV could be an effective, naturally adjuvanting, time-release antigen delivery system for immunologically relevant Chlamydia vaccine antigens which are readily recognized by human immune sera.     

Ecological transition from natural forest to tea plantations: effect on the dynamics of malaria vectors in the highlands of Cameroon

From October 2002 to September 2003, an entomological survey was carried out in a rural forested fringed village in the highlands of Mount Cameroon region to determine the temporal dynamics of the anopheline population and the intensity of malaria transmission. A total of 2387 Anopheles spp. were collected, with A. funestus predominating (59.9%), followed by A. hancocki (24.4%) and A. gambiae s.l. (15.7%). Considerable differences were observed in the nocturnal biting cycles of parous mosquitoes, with peak activity in the latter part of the night. PCR revealed that all specimens of the A. funestus group were A. funestus s.s. and all specimens from the A. gambiae complex were A. gambiae s.s. of the S molecular form. Plasmodium falciparum sporozoite rates of 17.3% and 8.5% were recorded for A. funestus and A. hancocki, respectively, with an anthropophilic rate of 96.3%. A strong positive correlation (r = 0.996) was found between the human-biting rate and the entomological inoculation rate (EIR). Malaria transmission was very high and perennial, with an estimated annual EIR of 460.1 infective bites per person per year. These results confirm that in high agricultural activity areas, A. funestus can be by far the major malaria vector responsible for malaria transmission.     

Noise levels in an urban Asian school environment

Background noise is known to adversely affect speech perception and speech recognition. High levels of background noise in school classrooms may affect student learning, especially for those pupils who are learning in a second language. The current study aimed to determine the noise level and teacher speech-to-noise ratio (SNR) in Hong Kong classrooms. Noise level was measured in 146 occupied classrooms in 37 schools, including kindergartens, primary schools, secondary schools and special schools, in Hong Kong. The mean noise levels in occupied kindergarten, primary school, secondary school and special school classrooms all exceeded recommended maximum noise levels, and noise reduction measures were seldom used in classrooms. The measured SNRs were not optimal and could have adverse implications for student learning and teachers’ vocal health. Schools in urban Asian environments are advised to consider noise reduction measures in classrooms to better comply with recommended maximum noise levels for classrooms.     

Genetic biomonitoring of inhabitants exposed to uranium in the north region of Brazil

Aiming to assess the susceptibility of populations in the Brazilian Amazon region to ionizing radiation emitted from uranium, mutations frequencies in the DNA repair genes XRCC1 and XRCC3 and in the metabolic gene GSTM1 were evaluated. The XRCC1 allele frequencies for the 194Trp polymorphism in the municipalities of Monte Alegre, Prainha and Alenquer were, respectively, 0.12, 0.13 and 0.07, and for 399Gln polymorphism they were, respectively, 0.28, 0.30 and 0.32. Frequencies for GSTM1 gene deletion homozygotes were, respectively, 0.36, 0.31 and 0.40 for all municipalities. These frequencies are comparable to those described for Brazilian individuals from other regions of the country. Also, allele frequencies of XRCC3 241Met polymorphism of the Monte Alegre and Alenquer populations were 0.28 and 0.33, respectively. In conclusion, frequencies of important polymorphic features of cellular DNA repair and metabolic apparatus in the populations studied do not differ from those of populations in other regions of Brazil.     

Leptosome-entrapped leptospiral antigens conferred significant higher levels of protection than those entrapped with PC-liposomes in a hamster model

We prepared novel liposomes from total polar lipids of non-pathogenic Leptospira biflexa serovar Potac (designated leptosomes) and evaluated their vaccine delivery/adjuvant potential with novel protective antigens (Lp0607, Lp1118 and Lp1454) of L. interrogans serovar Pomona in a hamster model. The immune response induced by three individual antigens and protective efficacy were evaluated and compared to those induced by same antigens entrapped with PC-liposomes and E. coli lipid liposomes (escheriosomes). Four-week-old hamsters were immunized subcutaneously twice at a 3-week interval, bled at various time points to evaluate antibody response and sacrificed to isolate splenocytes for lymphocyte proliferation and cytokine profiles in response to recall antigen. For the challenge test, 10× MLD₅₀ (modified lethal dose 50%) of virulent L. interrogans serovar Pomona were administered intraperitoneally. Our results demonstrate that leptosome are better adjuvant than PC-liposomes as revealed by enhanced long term antibody response, lymphocyte proliferation and significant enhancement of both Th1 (IFN-γ) and Th2 (IL-4 and IL-10) cytokines. Additionally, leptosomes and escheriosomes induced significantly higher level of memory responses than PC-liposome did. Moreover, the novel leptosomal vaccine induced significantly higher levels of protection than those prepared with PC-liposomes as revealed by enhanced survival, reduced histopathological lesions in vital organs and reduced leptospiral load in kidneys. Taken together, the results of the present study clearly reveal that both leptosomes and escheriosomes have emerged as promising delivery vehicles/adjuvants that can be widely exploited with newly discovered antigens in future leptospira vaccines.