Prevalence of Schistosoma haematobium infection among inhabitants of Lowveld, Swaziland, an endemic area for the disease.

We carried out a parasitological survey of Schistosoma haematobium infection among the residents of Lowveld Siphofaneni, Swaziland, an area which is devoid of sanitation. Subjects with positive infection were confirmed by the detection of S. haematobium ova in their urine. The intensity of the infection was estimated by calculating the total number of S. haematobium ova present in 10 ml urine specimen (geometric mean intensity; GMI). Overall, the prevalence of S. haematobium infection was 6.1% (18/295) with a GMI of 20.7 (95% CI=9.1~32.2). Female (10.5%, 16/153) had significantly higher prevalence than that in male (1.4%, 2/142) (ORs=8.2, 95% CI=1.8- 36.2, P<0.01); conversely, male had higher GMI (60.0) than that (17.3) in female. The age group of ≤5 yrs (15.3%, 9/59) had significantly higher prevalence than that in age group of ≥19 yrs (2.6%, 3/115) (ORs=0.2, 95% CI=0.04-0.57, P<0.01). The highest GMI of 27.9 (95% CI=7.6~48.2) was also seen in age group of ≤5 yrs.

Seroprevalence of Toxocara canis infection among children in Swaziland, southern Africa.

As there appeared to be no data available on Toxocara canis infection in the children of Swaziland, a serological survey of T. canis infection was recently conducted among 92 children aged 3-12 years from rural slums in the low- and middle-veld. A child was considered seropositive if, in western blots based on the excretory-secretory antigens of larval T. canis, his or her serum gave a positive result when diluted 1 : 64. Forty-one (44.6%) of the children were found seropositive. There were no statistically significant differences in seroprevalence between the 49 boys and 43 girls investigated (46.9% v. 41.8%) or between the eight subjects aged 12 years and the 47 aged < or = 5 years (62.5% v. 38.3%); the corresponding odds ratios were 0.81 (95% confidence interval=0.36-1.86; P=0.62) and 2.69 (95% confidence interval=0.57-12.62; P=0.20), respectively. The 66 subjects from the middleveld were, however, significantly more likely to be seropositive than the 26 subjects from the lowveld (54.5% v. 19.2%; odds ratio=5.04, with a 95% confidence interval of 1.70-14.98; P<0.01). It seems likely that T. canis infection is common among the children who live in slums in Swaziland, particularly in the country's middleveld, probably as the result of poor hygiene and poor sanitation.

Estimating intracellular Ca2+ concentrations and buffering in a dendritic inhibitory hippocampal interneuron.

Calcium is known to regulate several phenomena like neuronal excitability and plasticity. Interestingly, the spatiotemporal profile of dendritic calcium depends on several processes, specific to each neuronal type. In this study, we investigated Ca(2+) buffering and action potential (AP)-evoked Ca(2+) signaling in the dendrites of anatomically identified oriens lacunosum-moleculare (O-LM) cells, a major type of dendrite-targeting interneurons in the hippocampal CA1 region, using a combination of whole-cell patch-clamp recording and fast Ca(2+) imaging in acute rat brain slices. Cells were loaded with fluorescent Ca(2+) indicators fura-2 or Oregon Green BAPTA-1 (OGB-1) via patch-clamping electrode, and the effect of fura-2 on AP-evoked dendritic Ca(2+) transients was determined by ratiometric Ca(2+) imaging. To estimate intracellular Ca(2+) concentrations ([Ca(2+)](i)) and endogenous Ca(2+)-binding ratio (kappa(s)) in the proximal dendrite, fluorescence signals were converted into [Ca(2+)](i) using the ratioing method and were analyzed on the basis of the "single compartment model." Resting [Ca(2+)](i) was 22+/-5 nM and the build-up of [Ca(2+)](i) during a single AP was up to 656+/-226 nM. Analysis of Ca(2+) transients revealed that O-LM cells have a relatively low endogenous Ca(2+)-binding ratio (kappa(s)): the kappa(s) was 20+/-8 estimated during fura-2 loading and 27 estimated under steady-state fura-2 concentrations, respectively. To further examine the spatial profile of dendritic Ca(2+) transients, we measured somatic AP-evoked Ca(2+) transients beyond proximal dendrites using OGB-1. Dendritic Ca(2+) transients evoked by single APs or AP trains are not limited to regions close to the soma. The amplitude and decay of [Ca(2+)](i) associated with backpropagating APs are relatively independent of the distance from the soma. In sum, O-LM cells exhibit low endogenous Ca(2+)-binding ratios and relatively distance-independent Ca(2+) dynamics in the dendrites. These special features of Ca(2+) signaling in O-LM cells may have important functional implications for both normal and pathological conditions.

Blood-brain barrier impairment with enhanced SP, NK-1R, GFAP and claudin-5 expressions in experimental cerebral toxocariasis.

Infection by Toxocara canis in humans may cause cerebral toxocariasis (CT). Appreciable numbers of T. canis larvae cross the blood-brain barrier (BBB) to invade the brain thus causing CT. In the present studies, we evaluated the BBB permeability and BBB injury as assessed by the cerebral Evans blue (EB) concentration as well as by pathological changes and glial fibrillary acidic protein (GFAP) expression in T. canis -infected mice monitored from 3 days (dpi) to 8 weeks post-infection (wpi). The vasodilation neuropeptides, the expressions of substance P (SP) and its preferred binding neurokinin-1 receptor (NK-1R) as well as claudin-5 of tight-junction proteins associated with BBB impairment were also assessed by Western blotting and reverse-transcriptase polymerase chain reaction. Results revealed that BBB permeability increased as evidenced by a significantly elevated EB concentration in brains of infected mice. BBB injury appeared due to enhanced GFAP protein and mRNA expressions from 4 to 8 wpi. Leukocytes might have been unrelated to BBB impairment because there was no inflammatory cell infiltration despite T. canis larvae having invaded the brain; whereas markedly elevated SP protein and NK-1R mRNA expressions concomitant with enhanced claudin-5 expression seemed to be associated with persistent BBB impairment in this experimental CT model.

Sero-epidemiology of Toxocara canis infection among aboriginal schoolchildren in the mountainous areas of north-eastern Taiwan.

A sero-epidemiological study of Toxocara canis infection was conducted among Atayal schoolchildren (aged 7-12 years) residing in the mountainous areas of north-eastern Taiwan. The 73 children investigated were each checked for anti-Toxocara IgG, in ELISA based on the larval excretory-secretory antigens of T. canis larvae. A short, self-administered questionnaire was then used to collect relevant information from each subject, including data on the keeping of dogs, playing in soil, eating raw vegetables, and whether the subjects normally washed their hands before eating. Once the seropositive children had been identified, odds ratios (OR), with their corresponding 95% confidence intervals (CI) and P-values, were calculated for each potential risk factor. When diluted 1:64, sera from 42 (57.5%) of the children gave a positive result in the ELISA, indicating that these 42 children were seropositive for T. canis infection. Seropositivity did not appear to be associated with the age or gender of the subject, the eating of raw vegetables, or the regular failure to wash hands prior to a meal. Compared with the other subjects, however, those who admitted living in a household where dogs were kept (OR = 3.79; CI = 1.23-11.69; P = 0.02) or playing in soil (OR = 3.00; CI = 1.10-8.16; P = 0.03) appeared at increased risk of seropositivity.

Orally administrable enterotoxigenic Escherichia coli vaccine encapsulated by ethylcellulose powder dispersion.

To overcome the limitations of injection administration to vaccinate neonatal piglets against diarrheal disease, an oral vaccine needs to be developed. Enteric microspheres of oral vaccines were developed by a co-spray drying process based on formalin-inactivated enterotoxigenic Escherichia coli antigens with various encapsulating materials. The encapsulating efficiencies of ECN7m, ECN14m and ECN22m (vaccine microsphere formulations) tested by extraction procedure are high, more than 85%. To assess enteric characteristics, an in vitro dissolution test was performed with microspheres. Formulations with ethylcellulose ECN14m and ECN22m allow controlled release in a neutral or basic environment and resisted acid damage. In all cases, 95% of the E. coli protein was released within 2 h at pH 6.8-7, but there was no release at pH 1.5-2. However, ECN7m was less acid-resistant and had lower release at low pH. In animal immunization tests, oral immunization with microspheres of formulations ECN14 and ECN22m effectively evoked both systemic IgG and mucosal IgA responses against E. coli whole cell antigens in mice. In the mice challenge test, orally administrable ECNm14 (12 mg) or ECN22m (12.6 mg) vaccine (i.e., encapsulating 3.0x10(9) cfu inactive bacterial mass) provided good protection from infection in animals.

Protective effects of oral microencapsulated Mycoplasma hyopneumoniae vaccine prepared by co-spray drying method.

The efficacy of Mycoplasma hyopneumoniae oral vaccine was investigated in microsphere dosage form. A co-spray drying process was used to apply an encapsulating material, Eudragit L30 D-55, to microspheres containing Mycoplasma hyopneumoniae antigens. The microspheres were generally effective (>93%) with protein release at pH 7.4, but almost none were released at pH 1.2, for 3 hr in an in vitro dissolution test. An SPF-swine model was used to evaluate the effectiveness of the microspheres as an oral vaccine, and the related immune responses. The serum's systemic IgG against M. hyopneumoniae was evoked by ELISA analysis, after a 2nd immunization of all pigs. The vaccinated groups' mean lesion score was significantly lower after the Mycoplasma hyopneumoniae challenge than that of the nonvaccinated/challenged groups (P<0.05). This study strongly suggests that the oral microspheres vaccine prepared by a co-spray drying method can provide effective protection against M. hyopneumoniae infection in pigs.

PCR amplification of the Salmonella typhimurium fimY gene sequence to detect the Salmonella species.

This study evaluated the suitability of fimY gene amplification by PCR as an effective means of detecting Salmonella species. Although fimY gene of Salmonella typhimurium is involved in regulating type 1 fimbrial expression, the amino acid sequence of FimY shares very little homology with other known prokaryotic proteins in the GenBank database. Therefore, fimY is a promising target gene to detect the presence of Salmonella species. Herein, two primers internal to the fimY gene of S. typhimurium are used to investigate the distribution of the fimY homologous sequence among 45 Salmonella serovars and 20 non-Salmonella species by using PCR. Experimental results indicated that only Salmonella species possessed the fimY homologous sequence, subsequently generating the specific 526-bp DNA fragments. The sensitivity of the fmY-specific primer set was demonstrated on a Salmonella-free swab sample from a pork carcass surface, which was then artificially contaminated with different concentrations of S. typhimurium. A combining of pre-enrichment step in buffered peptone water and PCR amplification of fimY allowed the detection of S. typhimurium at the concentration of 3.4 x 10(0) CFU/ml from the swab sample. With an additional enrichment step in Rappaport-Vassiliadis (RV) broth, this procedure can also detect pork carcass surface naturally contaminated with Salmonella species in a slaughterhouse. Results in this study demonstrate that fimY is unique to Salmonella species and is an appropriate PCR target for detecting these microorganisms.

In vivo and in vitro comparisons of spray-drying and solvent- evaporation preparation of microencapsulated Mycoplasma hyopneumoniae for use as an orally administered vaccine for pigs.

OBJECTIVE: To evaluate the efficacy of an orally administered vaccine of Mycoplasma hyopneumoniae that was prepared by spray drying or solvent evaporation. ANIMALS: Thirty 6-week-old, crossbred, specific-pathogen-free (SPF) pigs. PROCEDURE: Pigs were randomly allocated into 5 groups and housed in an SPF facility. Pigs in 2 groups (groups AQ and CAP) were fed M hyopneumoniae enteric-coated vaccine on days 0, 10, and 20. A third group (group IM) received an IM injection of M hyopneumoniae vaccine with aluminium hydroxide as an adjuvant on days 0, 10, and 20. The last 2 groups (non-vaccinated-challenged [NV-C] and nonchallenged [NC]) were fed a sham treatment. All 24 pigs in groups AQ, CAFP IM, and NV-C were challenge exposed with 5 ml of a 10% pneumonic lung suspension administered on day 40 via intubation of the trachea. All pigs were slaughtered and the lungs removed and examined for lesions on day 68. RESULTS: In vitro studies indicated that these 2 microencapsulation techniques formed an effective shell and protected mycoplasmal antigen from gastric acid. Results of inoculation and challenge tests indicated that microencapsulated M hyopneumoniae were sufficiently potent to induce an immune response and provide good protection. CONCLUSIONS AND CLINICAL RELEVANCE: Orally administered microencapsulated M hyopneumoniae vaccines induced an immune response and reduced the severity of lung lesions in challenge-exposed pigs. Results suggest that this novel method can be applied to other antigens, because the spray-drying process yielded an orally administered M hyopneumoniae vaccine that induced a good immune response.

Seroepidemiologic survey of Dirofilaria immitis infection among domestic dogs in Taipei City and mountain aboriginal districts in Taiwan (1998-1999).

To estimate the seroprevalence of Dirofilaria immitis infection in domestic dogs in Taiwan, we utilized a commercial ELISA kit (Snap, IDEXX, USA) for detecting circulating antigens released by adult female worms. Serum specimens of 664 domestic dogs sampled from Taipei City in northern Taiwan and 14 mountain aboriginal districts in eastern Taiwan were screened for D. immitis antigens. Multivariate-adjusted odds ratios (ORs) with their 95% confidence intervals (CIs) were estimated by multiple logistic regression analysis. A total of 89 subjects were antigen-positive, giving a seroprevalence of 13.4%, of which the seroprevalence in Taipei City and mountain aboriginal districts were 13.8 and 12.1%, respectively. The mean overall seropositive rates were 6.3% in 1-3-year-old age group, 14.1% in 3-6-year-old age group and 23.7% in the > or =6-year-old age group. The older the age, the higher the seroprevalence (OR=4.6, 95% CI=2.4-9.0 for the > or =6-year-old age group versus 1-3-year-old age group, P<0.001) for all the dogs in the present study. Moreover, seroprevalence was not different between female and male dogs in either Taipei City or mountain aboriginal districts (P>0.05). Also, no significant difference in seroprevalence among dogs between the two geographical areas was found (P>0.05). In the logistic regression analysis, the seroprevalence of D. immitis remained significantly increased with age after multivariate adjustment in the present study. To our knowledge, this is the first report on the status of D. immitis infection in domestic dogs in Taipei City and mountain aboriginal districts in Taiwan to date.

Toxoplasma gondii infection: relationship between seroprevalence and risk factors among inhabitants in two offshore islands from Taiwan.

A seroepidemiological study of toxoplasmosis among inhabitants of Penghu Island and Kinmen Island offshore of Taiwan was performed using the latex agglutination test from July 1999 to June 2000. In order to determine risk factors for Toxoplasma gondii (T. gondii) infection, the effects of a history of eating raw/undercooked meats and raising pets were focused on using a self-administrated questionnaire. The seroprevalence (28.2%; 190/673) in Kinmen Island was significantly higher than that (2.71%; 8/293) in Penghu Island (P < 0.001). A significant difference in seroprevalence between both sexes was found in Kinmen Island (P < 0.05), but not in Penghu Island. The results of multiple logistic regression analysis showed that the older the age, the higher the OR in both Islands, yet a significant difference in seroprevalence between children and adults or the elderly was observed in Kinmen Island (P < 0.001). Moreover, those who had histories of raising cats or eating raw/undercooked meats seemed to have greater opportunities to become infected with T. gondii (OR = 2.9, 95% CI = 1.9-4.5, P < 0.001; OR = 1.5, 95% CI = 1.1-2.1, P < 0.05). In Penghu Island, a significant association between seroprevalence and a history of raising cats was also observed (OR = 4.6, 95% CI = 1.1-20.1, P < 0.05). Furthermore, workers, farmers, and fishermen seemed to be more susceptible to T. gondii infection than students in Kinmen Island.

A recombinant chimera composed of repeat region RR1 of Mycoplasma hyopneumoniae adhesin with Pseudomonas exotoxin: in vivo evaluation of specific IgG response in mice and pigs.

Using the binding and translocation domain of Pseudomonas exotoxin A [domain III deleted PE termed PE(DeltaIII)] as a vehicle, this study characterized and evaluated a novel application of PE toxin in Mycoplasma hyopneumoniae adhesin used as an immunogen. PCR and sequence analysis revealed that 16 copies of AAKPV(E) in tandem repeat region 1 (RR1) of M. hyopneumoniae 97kDa adhesion were successfully fused to the downstream of PE(DeltaIII) to create a subunit vaccine, i.e. PE(DeltaIII)-RR1. This chimeric protein, over-expressed in inclusion bodies of E. coli BL21(DE3)pLysS, was characterized by a monoclonal antibody (MAb) F2G5 prepared against RR1 of the 97kDa adhesin and was readily purified. The data indicated that the epitope recognized by MAb F2G5 was located in the structure of PE(DeltaIII)-RR1. Using ELISA and Western blot analyses, the specific IgG immune response against RR1 and whole adhesin in mice immunized with PE(DeltaIII)-RR1 was found more marked than that in mice immunized with the M. hyopneumoniae whole cells. Similarly, PE(DeltaIII)-RR1 also stimulated a remarkable IgG response against RR1 in pigs compared to that in pigs immunized with the conventional M. hyopneumoniae vaccine. The PE(DeltaIII)-RR1 would be potentially useful for the future development of a M. hyopneumoniae adhesin vaccine.

Release characteristics of microspheres prepared by co-spray drying Actinobacillus pleuropneumoniae antigens and aqueous ethyl-cellulose dispersion.

Using formalin inactivated Actinobacillus pleuropneumoniae antigens and aqueous ethylcellulose dispersions, microspheres of oral vaccines were developed by a co-spray drying process. The present study attempted to determine whether the dosage formulations of microspheres could form enteric matrices. To assess the enteric characteristics, an in vitro dissolution test was performed with the AQ6-AP microspheres; 95% of the A. pleuropneumoniae protein was released within 3 h at pH 7, but there was no release at pH 1.5. The scanning microscopy revealed that the surface structure of AQ6-AP microspheres became porous at neutral pH. The SDS-PAGE analysis showed that the release rate of proteins from the microspheres was pH dependent not only for the AQ6-AP formulation but also when antigens of A. pleuropneumoniae were replaced with porcine serum. The results suggest that the A. pleuropneumoniae antigens were entrapped in the AQ6 microspheres under the acidic conditions. In a mouse model, oral immunization with AQ6-AP microspheres containing A. pleuropneumoniae evoked systemic IgG and mucosal IgA responses against A. pleuropneumoniae antigens. Thus, the present method may further provide an opportunity to develop oral vaccines and mucosal immunity.

Entropy-driven binding/partition of amino acids/dipeptides to stratum corneum lipid vesicles.

This study employed large unilamillar vesicles composed of purchased stratum corneum lipids to investigate the binding/partition of amino acids/dipeptides to stratum corneum lipid vesicles. The partition coefficients of amino acids/dipeptides between the stratum corneum lipid vesicles and the acetate buffer were determined by HPLC. In addition, the binding/partition enthalpy of amino acids/dipeptides with the stratum corneum lipid vesicles was derived by directly measuring the binding/partition heat with isothermal titration calorimetry. According to the binding/petition Gibbs free energy and the binding/partition enthalpy, all the binding/partition of amino acids/dipeptides with the stratum corneum lipid vesicles is endothermic, implying an entropy-driven binding/partition. Also, the equilibrium binding/partition results demonstrate that the partition coefficients of amino acids/dipeptides do not correlate with the transdermal permeability. This finding suggests that either the interaction between the penetrants and the lipid bilayer between corneocytes may not be a determining step or that the paracellular path is not a dominant route of transdermal penetration.

A target-specific chimeric toxin composed of epidermal growth factor and Pseudomonas exotoxin A with a deletion in its toxin-binding domain.

We have fused the epidermal growth factor (EGF) to the amino terminus of Pseudomonas exotoxin A (PE) to create a cytotoxic agent, designated EGF-PE, which preferentially kills EGF-receptor-bearing cells. In this study, we analyzed the effect of the Ia domain, the binding domain of PE on the cytotoxicity of EGF-PE towards EGF-receptor-bearing cells and tried to develop a more potent EGF-receptor-targeting toxin. EGF-PE molecules with sequential deletions at the amino terminus of PE were constructed and expressed in E. coli strain BL21(DE3). The cytotoxicity of these chimeric toxins was then examined. Our results show that the amino-terminal and carboxy-terminal regions of the Ia domain of PE are important for the cytotoxicity of a PE-based targeting toxin. To design a more potent PE-based EGF-receptor-targeting toxin, a chimeric toxin, named EGF-PE(delta 34-220), which had most of the Ia domain deleted but retained amino acid residues 1-33 and 221-252 of this domain, was constructed. EGF-PE(delta 34-220) has EGF-receptor-binding activity but does not show PE-receptor-binding activity and is mildly cytotoxic to EGF-receptor-deficient NR6 cells. As expected, EGF-PE(delta 34-220) is a more potent cytotoxic agent towards EGF-receptor-bearing cells than EGF-PE(delta 1-252), where the entire Ia domain of PE was deleted. In addition, EGF-PE(delta 34-220) was shown to be extremely cytotoxic to EGF-receptor-bearing cancer cells, such as A431, CE81T/VGH, and KB-3-1 cells. We also found that EGF-PE(delta 34-220) was highly expressed in BL21(DE3) and could be easily purified by urea extraction. Thus, EGF-PE(delta 34-220) can be a useful cytotoxic agent towards EGF-receptor-bearing cells.

Effects of dietary energy intake by gilts and heat stress from days 3 to 24 or 30 after mating on embryo survival and nitrogen and energy balance.

Bred gilts were used in two experiments (n = 140) to study the interaction and main effects of dietary energy intake and heat stress on embryo survival and nitrogen (N) and energy balance from d 3 to slaughter on d 30 (Exp. 1) or d 24 (Exp. 2) after mating. In both experiments, the ME treatments were 5.4 or 8.1 Mcal of ME/(gilt.d). Temperatures were either a constant thermoneutral of 23 +/- 1 degrees C or a heat stress regimen, making four treatments in a 2 x 2 factorial arrangement. Gilts were allotted directly to one of the treatments on d 3 after mating. In Exp. 1, the 24-h cyclic heat stress regimen consisted of an increase from 25 degrees C at 0800 to 34 degrees C at 1400, 34 degrees C from 1400 to 1700, then a decline to 25 degrees C at 2000, with 25 degrees C until 0800. In Exp. 2, heat stress was constant at 33 +/- 1 degrees C. No energy x temperature treatment interactions occurred (P > .2 to .8) for the variables measured in Exp. 1 and 2. Heat stress reduced (P < .05 or .01) diet and ME intake/day, ME retained/day, and ME/GE compared with the thermoneutral controls in Exp. 1 and 2. Heat stress reduced (P < .05) N digestibility in Exp. 1 and reduced (P < .01) N retained/day and N retention/N intake in Exp. 2. Rectal temperature and respiration rate were increased (P < .01) by heat stress in Exp. 1 and 2.(ABSTRACT TRUNCATED AT 250 WORDS)