Effect of community participation on household environment to mitigate dengue transmission in Thailand.

Due to the absence of dengue vaccination, vector control is the only measure to prevent dengue outbreaks. The key element of dengue prevention is to eliminate vector habitats. Clean household environment, preventive behaviors of household members and community participation in dengue prevention and control are key successful elements. This study aimed to investigate the associations between environmental factors, dengue knowledge, perception and preventive behaviors of household and collaboration of community members and household risk of dengue by using mixed methods. One dengue epidemic province was selected from each region of Thailand including Bangkok. Two districts, one from the highest and another from the lowest dengue incidence areas, were selected from those provinces. The household leaders, community members, and local authorities in highest dengue incidence areas were interviewed by using questionnaire and through group interviews. The environment of each selected household was observed. Of 4,561 households, 194 were reported having dengue case(s) in the past year and that outdoor solid waste disposal significantly influenced household risk of dengue (OR=1.62; 95% CI=1.16-2.29). In contrast, having gardening areas reduced dengue risk at household level by 32%. High level of community participation in dengue prevention and control in uninfected areas and the information from local authorities and community members reconfirmed that community participation was the key factor against dengue outbreaks. Sustainable process of encouraging community members to eliminate vector breeding sites such as outdoor solid waste disposal is likely to lead to an achievement in dengue prevention and control.

Imported bancroftian filariasis: diethylcarbamazine response and benzimidazole susceptibility of Wuchereria bancrofti in dynamic cross-border migrant population targeted by the National Program to Eliminate Lymphatic Filariasis in South Thailand.

The implementation on the Thailand-Myanmar border of annual mass drug administration (MDA) of a single 6 mg/kg dose of diethylcarbamazine (DEC) plus 400mg albendazole, part of the National Program to Eliminate Lymphatic Filariasis (PELF), has been challenging. In particular, chain migration of cross-border Myanmar workers at risk for nocturnally periodic Wuchereria bancrofti infection can lead to imported bancroftian filariasis (IBF) in Thailand. IBF is targeted for multiple-dose MDA with 300 mg DEC, in addition to what is recommended by the World Health Organization (WHO). The dynamic Myanmar migrants in Phang-nga, southern Thailand were sampled to test whether the responsible W. bancrofti has a genetic predisposition of benzimidazole exposure, and IBF exhibits DEC susceptibility. The long-term migrants had more access to DEC. IBF in W. bancrofti antigenemic (microfilaremic vs. amicrofilaremic) short-term migrants exhibited susceptibility to a 300-mg single-dose DEC treatment. During the course of a 3-month follow-up, antigenemia was significantly reduced, but microfilaremia was fluctuated. Surprisingly, a newly recognized Mansonella infection co-existing among W. bancrofti-affected Myanmar migrants elicited microfilaremia clearance within a month after treatment. As a result of the presence of genetically stable W. bancrofti beta-tubulin (Wbtubb) gene responsible for benzimidazole susceptibility, IBF did not possess a genetic predisposition for benzimidazole exposure. Point mutations at positions Phe167Tyr and Phe200Tyr were not detected by Wbtubb locus-specific nested PCR and sequencing. This study has the potential to help guide not only the Thai/Myanmar PELF surveillance and monitoring of mass treatment impacts on W. bancrofti, but also the other endemic countries allied with the Global Program to Eliminate Lymphatic Filariasis (GPELF).

Toxicity of chitinase-producing Bacillus thuringiensis ssp. kurstaki HD-1 (G) toward Plutella xylostella.

One-hundred fifty isolates of Bacillus thuringiensis were tested for their ability to produce chitinase using colloidal chitin agar as the primary plating medium. Of 14 strains that produced chitinase, B. thuringiensis ssp. kurstaki HD-1(G) was identified as the highest chitinase producer and selected for further study. This bacterium produced the highest amount of chitinase (19.3 mU/ml) when it was cultivated in nutrient broth supplemented with 0.3% colloidal chitin on a rotary shaker (200 rpm) at 30 degrees C for 2 days. The toxicities of B. thuringiensis ssp. kurstaki HD-1(G) and B. thuringiensis ssp. kurstaki wa-p-2, a chitinase nonproducer, were assayed toward Plutella xylostella (diamondback moth) larvae, resulting in LC(50)'s of 4.93 x 10(4) and 1.32 x 10(5) spores/ml, respectively. If the culture broth from B. thuringiensis ssp. kurstaki HD-1(G) was used as the suspending liquid instead of phosphate buffer, their LC(50)'s were reduced to 6.23 x 10(3) and 7.60 x 10(4) spores/ml, respectively. The histopathological changes of the midgut epithelial cells of diamondback moth larvae were compared after feeding on B. thuringiensis ssp. kurstaki HD-1(G) with and without the presence of supernatant containing chitinase under light microscopy and transmission electron microscopy. The midgut epithelial cells of larvae fed for 30 min in the presence of chitinase, with or without spores and endotoxin crystals, appeared more elongated and swollen than those of the control larvae. A number of different cellular changes such as extensive cellular disintegration and appearance of numerous vacuoles were observed from the larvae fed on B. thuringiensis ssp. kurstaki HD-1(G) supplemented with supernatant containing chitinase. Thus increased toxicity and changes in epithelial cells were correlated with the presence of chitinase but this was not distinguished from the possible presence of vegetative-stage insecticidal proteins.

Purification and characterization of chitinase from Bacillus circulans No.4.1.

Bacillus circulans No.4.1 produced a high level of chitinase when cells were grown in tryptic soy broth supplemented with 0.3% colloidal chitin at 35 degrees C for 5 days. Purification was carried out by protein precipitation with 80% saturation ammonium sulfate, anion-exchange chromatography with DEAE-Sephacel, and gel filtration with Sephadex G-100, sequentially. The purified enzyme could be demonstrated as a single band on SDS-PAGE, estimated to be 45 kDa. This enzyme could hydrolyze colloidal chitin, purified chitin, glycol chitin, carboxymethyl-chitin (CM-chitin), and 4-methylumbelliferyl-beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)(2)]. The optimal conditions for this chitinase were pH 8.0 and 40 degrees C. The isoelectric point of the chitinase was 5.1. The amino acid composition of the purified chitinase was determined. The initial 20 amino acid residues of the N-terminal were found to be alanine (A), proline (P), tryptophan (W), asparagine (N), serine (S), lysine (K), glycine (G), asparagine (N), tyrosine (Y), alanine (A), leucine (L), proline (P), tyrosine (Y), tyrosine (Y), arginine (R), glycine (G), alanine (A), tryptophan (W), alanine (A), and valine (V). Knowledge of these properties of chitinase from B. circulans No. 4.1 should be useful in the development of genetically engineered Bacillus sp. as biopesticides.

Field trial of the ICT filariasis for diagnosis of Wuchereria bancrofti infections in an endemic population of Thailand.

The ICT Filariasis, a rapid card test format, which is based on qualitative detection by monoclonal antibody of the circulating antigen of Wuchereria bancrofti adult worm, is a new diagnostic test of choice for determining the infections under field conditions. By using clinical and recall techniques and microscopy (thick smear and capillary tube technique) as reference, we assessed the efficiency of the ICT card test in sera from 225 subjects living in W. bancrofti-endemic villages of Tak Province, Thailand, who were recruited during a cross-sectional community survey. The ICT card test gave a 20% antigen positive rate, while other tests gave lower positive rates of the same 5.8% by clinical and recall techniques and thick smear, and 5.3% by capillary tube technique, respectively. The ICT card test had a specificity of 100% when sera from microfilaremic subjects were positive, as when sera from W. bancrofti non-endemic subjects either with Brugia malayi microfilaremia or with other parasites, and those from normal controls were all negative by the test. When done in W. bancrofti microfilaremia sera, the ICT card test had a sensitivity of 100% using a microscopy as reference, and 84.6% when using clinical and recall techniques. However, the ICT card test was more sensitive than the others when done in endemic normal sera (14% positive). Such findings compared well with findings in endemic area of South America, suggested its usefulness to detect W. bancrofti infections in endemic area, especially on the Thai-Myanmar border.

Expression of chitinase-encoding genes from Aeromonas hydrophila and Pseudomonas maltophilia in Bacillus thuringiensis subsp. israelensis.

Fifty isolates of chitinase (Cts)-producing bacteria were collected from soil samples and tested for their ability to degrade chitin using colloidal chitin agar as the primary plating medium. The results indicated that three isolates could degrade chitin at high pH. Further studies also demonstrated that crude Cts preparations from Bacillus circulans (Bc) No. 4.1 could enhance the toxicity of Bacillus thuringiensis subsp. kurstaki (Bt-k) toward diamondback moth larvae. Thus, it might be useful to increase the toxicity of B. thuringiensis (Bt) toward target insects by introducing a Cts-encoding gene (cts) into Bt. To investigate the expression of cts in Bt, cloned cts from Aeromonas hydrophila (pHYA1) and Pseudomonas maltophilia (pHYB1, pHYB2 and pHYB3) were cloned into the shuttle vector pHY300PLK and transformed into Escherichia coli DH5 alpha using 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside (4-MUF GlcNAc) as the detecting substrate. The four plasmids were then introduced into B. thuringiensis subsp. israelensis (Bt-i) strain c4Q272 by electroporation. Various transformants harboring cloned cts were selected, and expression and stability of the plasmids in Bt were studied.

Biodegradation of 3-chlorobenzoate by Pseudomonas putida 10.2.

Pseudomonas putida 10.2, a 3-chlorobenzoate (3CBa)-degrading bacterium, was isolated from a soil sample obtained from an agricultural area in Chiang Mai, Thailand. This bacterium could degrade 2MM 3CBa very rapidly with the concomitant formation of chloride ion when grown in mineral salt-yeast extract medium. The presence of glucose, lactose and pyruvate in the medium reduced the capability of this bacterium to degrade 3CBa. Metabolites such as 3-chlorocatechol (3CC), catechol and cis,cis-muconic acid (muconate) could be detected in the growth medium or in cell suspensions when 3CBa was used as the substrate. Furthermore, when crude enzyme extract prepared from 3CBa-grown P. putida 10.2 was incubated with 3CC, catechol and muconate could be detected in the reaction mixtures. Thus, the biodegradation pathway of 3CBa by P. putida 10.2 was proposed to involve transformation of 3CBa to 3CC. The dehalogenation step is believed to involve removal of chloride from 3CC to form catechol, which is subsequently converted to muconate.

Plasmodium berghei: partial purification and characterization of the mitochondrial cytochrome c oxidase.

Mitochondria from a rodent malarial parasite (Plasmodium berghei) were successfully purified by differential centrifugation and 22% Percoll density gradient separation. The purified mitochondria from the erythrocytic stages of the parasite had a density of 1.05 and were found to be heterogeneous by transmission electron microscopy and rhodamine 123 fluorescence microscopy. Three marker enzymes, dihydroorotate dehydrogenase, cytochrome c reductase, and cytochrome c oxidase, were assessed during the organelle separation. Purification of cytochrome c oxidase was carried out from the purified mitochondria by using combination techniques of detergent solubilization and reduced cytochrome c-agarose affinity chromatography. The 560-fold purified enzyme with 3.6% yield was obtained and it had low catalytic efficiency with a kcat/Km of 5.9 x 10(-5) M-1 x min-1. The native form of the enzyme, determined by a gel filtration column on fast protein liquid chromatography, was found to be an oligomeric structure with a minimal molecular weight of 670 kDa. The malarial enzyme was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then compared to the enzyme obtained from host liver cells. These results suggested that the partially purified enzyme from the parasite was not different from its host mammalian cells. The importance of the enzyme in the erythrocytic phase of the parasite is discussed as a part of a simple electron transport system in mitochondrion linked to limited oxygen utilization and pyrimidine de novo biosynthesis.

Molecular cloning of the 130-kilodalton mosquitocidal delta-endotoxin gene of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus.

A 3.7-kilobase (kb) XbaI fragment harboring the cryIVB gene (L. Thorne, F. Garduno, T. Thompson, D. Decker, M. A. Zounes, M. Wild, A. M. Walfield, and T. J. Pollock, J. Bacteriol. 166:801-811, 1986) which encoded a 130-kilodalton (kDa) mosquitocidal toxin from a 110-kb plasmid of Bacillus thuringiensis subsp. israelensis 4Q2-72 was cloned into pUC12 and transformed into Escherichia coli. The clone with a recombinant plasmid (designated pBT8) was toxic to Aedes aegypti larvae. The fragment (3.7 kb) was ligated into pBC16 (tetracycline resistant [Tcr]) and transformed by the method of protoplast transformation into Bacillus sphaericus 1593 and 2362, which were highly toxic to Anopheles and Culex mosquito larvae but less toxic to Aedes larvae. After cell regeneration on regeneration medium, the Tcr plasmids from transformants (pBTC1) of both strains of B. sphaericus were prepared and analyzed. The 3.7-kb XbaI fragment from the B. thuringiensis subsp. israelensis plasmid was shown to be present by agarose gel electrophoresis and Southern blot hybridization. In addition, B. sphaericus transformants produced a 130-kDa mosquitocidal toxin which was detected by Western (immuno-) blot analysis with antibody prepared against B. thuringiensis subsp. israelensis 130-kDa mosquitocidal toxin. The 50% lethal concentrations of the transformants of strains 1593 and 2362 against A. aegypti larvae were 2.7 X 10(2) and 5.7 X 10(2) cells per ml, respectively. This level of toxicity was comparable to the 50% lethal concentration of B. thuringiensis subsp. israelensis but much higher than that of B. sphaericus 1593 and 2362 (4.7 X 10(4) cells per ml) against A. aegypti larvae.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotinylated probes for epidemiological studies of drug resistance in Salmonella krefeld.

A gene probe for ampicillin resistance and one for sulphonamide resistance were prepared to study the origin and the relation of multiple drug resistances in Salmonella krefeld. The resistance genes were cloned into the pACYC184 vector of Escherichia coli from a common plasmid of S. krefeld that encoded for resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, sulphonamide and tetracycline resistance. Restriction map analysis and deletion analysis of a recombinant plasmid (pACSS1) showed that the gene determining ampicillin resistance was located on a 1.34 and 1.12 kb PstI fragment, and that the gene for sulphonamide resistance was located on a 0.85 kb PstI fragment. These fragments were used as probes. Their specificity was tested by colony hybridization with various bacterial species, including sensitive and resistance S. krefeld isolates. Further study indicated that the ampicillin resistance gene probe reacted with the gene for TEM-1 beta-lactamase and that the gene probe for sulphonamide resistance reacted with the gene for type II dihydropteroate synthase. The two probes were sufficiently specific to allow study of the epidemiology of resistance in S. krefeld and other enteric bacteria.

Current biotechnological developments in Thailand.

Thailand is very much aware of the potential and the opportunities in biotechnology and has given the utmost effort into the development of biotechnology. In 1983, the government has set up the National Center for Genetic Engineering and Biotechnology (NCGEB). The center operates through a network of research institutes and laboratories in order to maximize and consolidate the limited resources of the country. The center also plays a key role in formulating policies and plans relating to biotechnology as well as in supporting and coordinating biotechnology research and development. A sum of U.S. $8.6 million has been allocated for an initial 5-year program for R D & E activities. The priority consideration is on utilizing various levels of biotechnology for improvement in agriculture, industrial productivity, health, and environment. To facilitate and strengthen the link between research institutions and the private sector, the high-level Science and Technology Development Board (STDB) was established in 1986, with an initial allocation of U.S. $2.9 million between 1986 to 1992 for biotechnology. At present, there are between 400 to 500 scientists and technologists with M.S. or higher degrees actively working in research and development (R & D) in biotechnology and engineering, mostly in universities and government research laboratories. It is expected that approximately 500 graduates with advanced degrees in biotechnology and related fields will be produced during the 5-year plan (1987 to 1991).

Occurrence of toxicity among protease, amylase, and color mutants of a nontoxic soy sauce koji mold.

A soy sauce koji mold, Aspergillus flavus var. columnaris Raper and Fennel (ATCC 44310), was treated with UV irradiation to obtain mutant strains possessing high protease activities, high amylase activities, and light-colored conidia. Selected mutant strains were tested for toxicity, and some were found acutely toxic to weanling rats, although all were negative for aflatoxin production.

Isolation of Alkaline and Neutral Proteases from Aspergillus flavus var. columnaris, a Soy Sauce Koji Mold.

Two different extracellular proteases, protease I (P-I), an alkaline protease, and protease II (P-II) a neutral protease, from Aspergillus flavus var. columnaris were partially purified by using (NH(4))(2)SO(4) precipitation, diethylaminoethyl-Sephadex A-50 chromatography, carboxymethylcellulose CM-52 chromatography, and Sephadex G-100 gel filtration. The degree of purity was followed using polyacrylamide gel electrophoresis. The activity of P-I was completely inhibited by 0.1 mM phenylmethylsulfonyl fluoride, and that of P-II was completely inhibited by 1 mM ethylenediaminetetraacetate. By using these inhibitors with extracts of wheat bran koji, the proportions of total activity that could be assigned to P-I and P-II were 80 and 20%, respectively. This compared favorably with activities estimated by using polyacrylamide gel electrophoresis slices (82 and 18%, respectively). Extracts from factory-run soybean koji gave comparable results. Both enzymes demonstrated maximum activity at 50 to 55 degrees C and only small changes in activity between pH 6 and 11. For P-I, activity was somewhat higher from pH 8.0 to 11.0, whereas for P-II it was somewhat higher from pH 6 to 9. In the presence of 18% NaCl, the activities of both P-I and P-II dropped by approximately 90 and 85%, respectively. P-I was inferred to possess aminopeptidase activity since it could hydrolyze l-leucyl-p-nitroanilide hydrochloride. P-II was devoid of such activity. The ramifications of the results for factory-produced soy sauce koji are discussed.

Problematic occurrence of tyrosine crystals in the thai soybean paste tao chieo.

In a Bangkok soy sauce factory that had recently converted to controlled inoculum and incubation for the koji stage of fermentation, a problem arose with unsightly white particles in the soybean paste condiment called tao chieo. This problem had not arisen during the previous history of the factory where the koji stage of the fermentation was uncontrolled. Microscopic examination of the particles showed that they were crystalline. Physical separation of the crystals followed by solvent extraction, recrystallization, chemical characterization, and spectroscopy showed that they consisted of tyrosine. Tao chieo prepared by using low- or high-protease strains of Aspergillus sp. indicated that the tyrosine crystals resulted from mold proteolysis of the soybean substrate. Addition of polyethylene glycol at the beginning of the moromi fermentation reduced the severity of crystal formation.

Isolation and analysis of molds from soy sauce koji in Thailand.

Five different isolates of Aspergillus and one of Mucor were compared with a Japanese commercial strain of Aspergillus oryzae for proteolytic activity on wheat bran substrate. One isolate of Aspergillus with superior protease production, identified as Aspergillus flavus var. columnaris, showed no detectable aflatoxin production on glutinous rice or soybean substrate. Preliminary tests using this fungus as a koji mold in a traditionally operated factory resulted in a soy sauce superior in quality to that usually produced.

Trehalose metabolism by Bacillus popilliae.

Trehalose was found to be utilized more readily than glucose for the growth of Bacillus popilliae NRRL B-2309MC. The pathway of degradation of trehalose was elucidated and found to differ from that reported for other organisms. Trehalase and trehalose phosphorylase activities could not be detected. Rather, trehalose was found to undergo phosphoenolpyruvate (PEP)-dependent phosphorylation, and the resulting trehalose 6-phosphate was cleaved by a phosphotrehalase to equimolar amounts of glucose and glucose 6-phosphate. The phosphotrehalase was purified 34-fold and shown to have a pH optimum of 6.5 to 7.0 and a K(m) for trehalose 6-phosphate of 1.8 mM. A mutant missing the phosphotrehalase failed to grow on trehalose but grew normally on other sugars. The mutant accumulated [(14)C]trehalose as [(14)C]trehalose 6-phosphate. Phosphorylation of trehalose by dialyzed extracts was at least 25 times faster with PEP than with adenosine 5'-triphosphate, and the phosphorylation activity was associated primarily with the particulate fraction. These data and the results of studies of [(14)C]trehalose uptake suggest that trehalose is transported into the cell as trehalose 6-phosphate by a PEP:sugar phosphotransferase system. Cell extracts of other strains of B. popilliae were also found to produce [(14)C]sugar phosphate from [(14)C]trehalose and to have phosphotrehalase activity.