Endocrine effects of environmental pollution on Xenopus laevis and Rana temporaria

To determine the capacity of sewage treatment work effluents to disrupt the endocrine system under semifield conditions, two amphibian species, Xenopus laevis and Rana temporaria, were exposed to the effluent of a regional sewage treatment plant in South Bavaria during larval development until completion of metamorphosis. Exposure was carried out in river water (Würm) as a reference, and a 1:12-mixture sewage effluent representing the real situation on the spot, and in a higher concentration of sewage using a 1:2 mixture. An accidental impact of industrial wastewater into the reference and dilution medium, Würm, which was caused by a spate in the respective area during the sensitive period of sex differentiation of amphibian larvae, is assumed to be responsible for the relatively high percentage of females observed by histological analysis in all treatment groups. All of these values were higher than those determined in controls exposed to artificial tap water in laboratory experiments conducted in a comparable study design. Sex ratios between species, revealed by the semifield study with decreasing portions of females from control to 1:12 to 1:2, were strongly correlated. Determination of biomarker-mRNA-levels in Xenopus liver using semiquantitative RT-PCR at the end of the experimental phase, when exposure regime has turned into the initially expected situation with the highest load of potential estrogens in the effluent, followed by 1:2 and 1:12 mixture, resulted in a significant increase of Vitellogenin-mRNA in female juveniles exposed to the highest portion of sewage, whereas expression of both androgen and estrogen receptor-mRNA showed no clear differences. The results concerning the induction of estrogenic biomarkers are in accordance with our findings for estrogen receptor binding of sample extracts from the Würm and sewage taken in parallel at the end of the experiment, when sewage extracts possessed a much higher ability to displace [3H]estradiol from the estrogen receptor than the ones extracted from the mixtures.     

Transmission-dynamic model to capture the indirect effects of infant vaccination with Prevnar (7-valent pneumococcal conjugate vaccine (PCV7)) in older populations

We developed an age-structured, transmission-dynamic, mathematical model to quantify the direct and indirect benefits of infant PCV7 vaccination. The model simulates the acquisition of asymptomatic carriage of Streptococcus pneumoniae and the development of fatal and non-fatal invasive pneumococcal disease (IPD) among vaccinated and unvaccinated individuals aged <2, 2–4, 5–17, 18–49, 50–64, and ≥65 years old. The model was parameterized using published US surveillance data, supplemented with data from published literature. The model predicts the observed incidence of IPD with good agreement and may be used to predict the impact of various vaccination strategies in the US or other populations yet to introduce PCV7.     

Using estimates of metal bioavailability in the soil and genetic variation of allozymes to investigate heavy metal tolerance in the earthworm Eisenia fetida (Oligochaeta)

In a recent study, we showed that the earthworm species Eisenia fetida, inhabiting an extremely high metal polluted compost heap on a wine farm, did not have elevated body loads of the metals but exhibited genotoxic tolerance when exposed to Cd in the laboratory (Voua Otomo and Reinecke, 2010). To unravel the mechanism behind the surprisingly low metal body burdens on one hand and genotoxic tolerance on the other hand, we investigated the estimated bioavailability of these metals (Cu, Zn, Pb and Cd) using sequential extraction methods with CaCl₂ and di-ethylene-triamine-pentaacetic acid (DTPA) and allozyme polymorphism in this field population, a laboratory control as well as a long-term Cd exposed population. The amounts of mobile (extracted with CaCl₂) and mobilizable (extracted with DTPA) metals in relation to the total (extracted with nitric acid) metals were all below 0.05% for all four metals, suggesting low availability for uptake. The low availability of these metals could not be explained by physico-chemical properties of soil but by the phenomenon of aging of the metals. There was no difference in allozyme frequency between metal tolerant and non-metal tolerant populations of E. fetida. This suggested that the tolerance found in earlier studies could be a mere physiological adaptation     

Characteristics of immune-competent amoebocytes non-invasively retrieved from populations of the sentinel earthworm Lumbricus rubellus (Annelida; Oligochaeta; Lumbricidae) inhabiting metal polluted field soils

Lumbricus rubellus is a cosmopolitan earthworm devoid of riboflavin-storing eleocytes; its immune competent coelomocytes are predominantly amoebocytes. Our aim was to determine whether amoebocyte cytometrics in L. rubellus are robust biomarkers for innate immunological responses to environmental pollutants. Investigations were conducted on populations inhabiting three unpolluted and five metalliferous (mainly Pb+Zn+Cd) habitats in the UK and Poland. Inter-population differences in worm mass and amoebocyte numbers did not consistently reflect soil or tissue metal concentrations. Flow cytometry indicated that autofluorescence of the amoebocytes differs between cells from the unpolluted and metal-polluted worms, and pinocytosis of neutral red by amoebocytes was lower (especially at 15 versus 60 min incubation) in worms from the polluted Poland site compared with the reference population. To conclude, amoebocyte cytometrics and functionality are potentially useful for environmental diagnostics; deployment is contingent on better understanding potential confounders.     

Mercury and lead: Assessing the toxic effects on growth and metal accumulation by Lemna minor

Lemna minor L. was used to investigate the toxic effects of Pb and Hg either alone or in various binary mixtures under the static test conditions. A full-strength Jacob culture medium was used for the long-term cultivation of duckweeds and the experiments. Tests were run for 4 and 7 days. The EC₅₀ values for Pb was estimated as 6.8±0.2 mg L⁻¹ and 5.5±0.1 mg L⁻¹ for a 4 and a 7-day test periods, respectively. Hg was much more toxic than Pb with the EC₅₀ of 0.64±0.03 mg L⁻¹ (4 days) and 0.48±0.02 mg L⁻¹ (7 days). The amounts of Pb uptake by the plants were determined by atomic absorption spectrometer (AAS). The cold vapor AAS-technique (CV-AAS) was used for Hg determination. The interactive effect between Pb and Hg on growth was evaluated as additive on the basis of statistical data analysis. However, an antagonistic interaction was observed on the metal accumulation efficiency.     

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.     

On the validity of self-reports and indirect reports to ascertain malaria prevalence in settings of hypoendemicity

Self-reports are commonly used to ascertain malaria prevalence in epidemiological studies conducted in settings where laboratory diagnosis is impractical. Most studies, however, do not use self-report per se, but indirect report, where one respondent provides responses for all household members. Studies also vary in terms of the time frame used for this ascertainment. The aim of our research was to determine the validity of self-report and indirect report in ascertaining malaria prevalence over six, eighteen and thirty-month time periods. Reports of malaria episodes collected through interviewer-administered questionnaires (193 self-reports, 614 indirect reports) were compared to microscopy-confirmed cases (principally Plasmodium vivax) registered at a government-run health post in the Peruvian Amazon. Test parameters were estimated using a Bayesian latent class approach for imperfect gold standards. Logistic regression analyses were performed to explore determinants associated with accurate responses. Malaria self-report for the thirty-month period prevalence had the highest sensitivity (91.0%). Specificity was maximized when malaria prevalence was measured over the last six months for both self-report (91.6%) and indirect report (96.7%). Accuracy was highest for the six-month period prevalence in both self-report (91.3%) and indirect report (96.4%). Respondents who were female, had more education, or who provided a report on behalf of a child ≤ 12 years of age, were generally more accurate. Both self-report and indirect report provides accurate estimates of malaria prevalence, especially over shorter periods of time. The choice between self-report and indirect report should ultimately depend on the research question and the target study population.     

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.     

Toxic effects of erythromycin, ciprofloxacin and sulfamethoxazole on photosynthetic apparatus in Selenastrum capricornutum

The effects of three antibiotics (erythromycin, ciprofloxacin and sulfamethoxazole) on photosynthesis process of Selenastrum capricornutum were investigated by determining a battery of parameters including photosynthetic rate, chlorophyll fluorescence, Hill reaction, and ribulose-1.5-bisphosphate carboxylase activity, etc. The results indicated that three antibiotics could significantly inhibit the physiological progress including primary photochemistry, electron transport, photophosphorylation and carbon assimilation. Erythromycin could induce acute toxic effects at the concentration of 0.06 mg L⁻¹, while the same results were exhibited for ciprofloxacin and sulfamethoxazole at higher than 1.0 mg L⁻¹. Erythromycin was considerably more toxic than ciprofloxacin and sulfamethoxazole and may pose a higher potential risk to the aquatic ecosystem. Some indices like chlorophyll fluorescence, Mg²⁺-ATPase activity and RuBPCase activity showed a high specificity and sensitivity to the exposure of erythromycin, and may be potentially used as candidate biomarkers for the exposure of the macrolide antibiotics.     

Sensitivity of Allium and Nicotiana in cellular and acellular comet assays to assess differential genotoxicity of direct and indirect acting mutagens

We have evaluated the extent of DNA damage induced by direct and indirect mutagens by cellular and acellular comet assays in two plant systems, Nicotiana tabacum (wild type tobacco) and Allium cepa (common onion). The objectives of this study were: (1) to generate dose-response curves for DNA migration values from root and shoot nuclei of A. cepa and N. tabacum treated with the direct acting mutagens, ethyl methanesulphonate (EMS), hydrogen peroxide (H₂O₂) and the indirect acting mutagen, cadmium chloride (CdCl₂), (2) to assess the differential response between isolated nuclei and nuclei of root and shoot and of both plants and (3) to examine the differences of sensitivity between direct and indirect acting mutagens by cellular and acellular comet assays. Similar sensitivities were evident in both plant systems to direct and indirect acting mutagens. The combination of cellular and acellular comet assays provided valuable insight to the mode of action of the genotoxicants used. The data obtained demonstrated the estimable capacity of the two plant systems to evaluate genotoxicity under different stress conditions and suggests Allium is a more desirable test system for rapid monitoring of genotoxicity.     

Affects of N-terminal variation in the SeM protein of Streptococcus equi on antibody and fibrinogen binding

The clonal Streptococcus equi causes equine strangles, a highly contagious suppurative lymphadenopathy and rhinopharyngitis. An important virulence factor and vaccine component, the antiphagocytic fibrinogen binding SeM of S. equi is a surface anchored fibrillar protein. Two recent studies of N. American, Japanese and European isolates have revealed a high frequency of N-terminal amino acid variation in SeM of S. equi CF32 that suggests this region of the protein is subject to immunologic selection pressure. The aims of the present study were firstly to map regions of SeM reactive with convalescent equine IgG and IgA and stimulatory for lymph node cells and secondly to determine effects of N-terminal variation on the functionality of SeM. Variation did not significantly affect fibrinogen binding or susceptibility of S. equi to an opsonic equine serum. Linear epitopes reactive with convalescent IgG and mucosal IgA were concentrated toward the conserved center of SeM. However, IgA but not IgG from every horse reacted with at least one peptide that contained variable sequence. Lymph node cells (CD4+) from horses immunized with SeM were strongly responsive to a peptide (αα36–138) encoding the entire variable region. SeM (CF32) specific mouse Mab 04D11 which reacted strongly with this larger peptide but not with shorter peptides within that sequence reacted strongly with whole cells of S. equi CF32 but only weakly with cells of any of 14 isolates of S. equi expressing different variants of SeM. These results in combination suggest that N-terminal variation alters a conformational epitope of significance in mucosal IgA and systemic T cell responses but does not affect antibody mediated phagocytosis and killing.     

Bottleneck effects on vaccine-candidate antigen diversity of malaria parasites in Thailand

A number of cell surface antigens of the infective stages of malaria parasites (genus Plasmodium) have been proposed as vaccine candidates, but high levels of polymorphism at the loci encoding these antigens are problematic for vaccine effectiveness. In order to test for the effects of anti-malarial control measures (including drugs and vector control) on polymorphism at antigen-encoding loci, we analyzed sequences of four antigen-encoding loci from P. vivax and two from P. falciparum collected in 2006–2007 from two areas of Thailand: (1) the NW, where malaria cases have remained high until recently; and (2) the South, where control measures have caused a dramatic decline in numbers of cases since 1990. Polymorphism in non-repeat regions of antigen-encoding loci was dramatically reduced in the South compared to the NW. These results suggest a two-pronged strategy for malaria eradication: (1) strenuous non-vaccine control measures that will cause a severe population bottleneck in the parasite; and (2) a subsequent local vaccine focused on one or a few locally occurring alleles at antigen-encoding loci.     

Two-generation effects of the chitin synthesis inhibitor, teflubenzuron, on the aquatic midge Chironomus riparius

Teflubenzuron, a chitin synthesis inhibitor was used in a two-generation test with Chironomus riparius (Insecta) to assess effects over a full life cycle from the first-instar larvae in the parental (P) generation until emergence in the subsequent F1 generation. Sediment spiked with teflubenzuron ranging from 10 to 390.6 μg/kg sediment dry weight (dw) was used. EC₅₀-values for fecundity and fertility were 112.7 and 74.5 μg/kg dw, respectively. Significant adverse effects were observed compared to the solvent control for emergence rate (P<0.01) and also for developmental rate. No observed effect concentrations values were lower for emergence rate in the F1 generation (62.5 μg/kg dw) than in the P generation (100 μg/kg dw), demonstrating that the F1 generation was more affected than the P generation.Thus, this two-generation test may help detecting population level effects as an amendment to the risk assessment for chronic exposures to endocrine disrupting compounds.     

Efficacy and side effects of praziquantel treatment in a highly endemic Schistosoma mansoni focus at Lake Albert, Uganda

The aim of the study was to assess the efficacy and side effects following single and repeated (6 weeks apart) praziquantel treatment (40 mg/kg) in a Schistosoma mansoni-endemic focus with long-standing transmission at Lake Albert in Uganda between December 1996 and January 1997. The results were based on 482 individuals, randomly representing all age and both gender groups. The cure rate following the first and second treatments was 41.9% and 69.1%, respectively. The cure rate was higher in adults than in children, irrespective of intensity of infection. In addition, the cure rate declined markedly with increasing intensity of infection. The reduction in intensity of infection was marked, being 97.7% and 99.6% after the first and second treatments, respectively. A pre- and post-treatment symptom questionnaire revealed a broad range of side effects, including abdominal pain and diarrhoea. However, no serious or long-lasting complications affecting compliance were observed. The marked reductions in faecal egg excretion and the acceptable level of side effects point to a single praziquantel treatment (40mg/kg) as the strategy of choice in such a highly endemic S. mansoni focus.     

Construction and evaluation of a live vaccine against Edwardsiella tarda and Vibrio harveyi: laboratory vs. mock field trial

Edwardsiella tarda and Vibrio harveyi are Gram-negative bacterial pathogens that affect a wide range of cultured fish. In previous studies, we have reported an E. tarda live vaccine ATCC15947 and a V. harveyi subunit vaccine DegQ. On the basis of these studies, in the present study we developed a cross protective vaccine against both E. tarda and V. harveyi by constructing a recombinant ATCC15947, Et15VhD, that expresses and secrets V. harveyi DegQ as a soluble antigen. Laboratory studies in a turbot (Scophthalmus maximus) model showed that Et15VhD elicited significant protections against E. tarda and V. harveyi when administered via intraperitoneal injection, oral feeding, immersion, and oral plus immersion, respectively. Microbiological analysis indicated dissemination and transient colonization of Et15VhD in fish tissues following vaccination. Since, compared to injection, oral plus immersion is a practically more acceptable vaccination procedure in aquaculture, we conducted a mock field trial to further examine the potential of Et15VhD as an oral plus immersion vaccine. The results showed that during the period before artificial bacterial challenge, mortality was observed in both the vaccinated group and the control group; however, the mortality of Et15VhD-vaccinated fish was significantly lower than that of the control fish. Following experimental challenge with E. tarda and V. harveyi at one and two months post-vaccination, Et15VhD-vaccinated fish exhibited dramatically increased survival rates compared to control fish. Serum antibody analysis indicated specific antibody production in Et15VhD-vaccinated fish. Taken together, these results demonstrate that Et15VhD induces strong protective immunity against E. tarda and V. harveyi under both laboratory and mock field conditions, which suggests a potential for Et15VhD to be used in aquaculture.