Experimental assessment of houseflies as vectors in avian influenza subtype H5N1 transmission in chickens.

In this study, laboratory-reared houseflies were experimentally exposed to the high pathogenicity avian influenza virus (HPAI) subtype H5N1 virus to evaluate the houseflies as vectors in HPAI-H5N1 virus transmission in chickens. One hundred and fifty houseflies (Musca domestica L.) were equally allocated into three groups. Groups 2 and 3 were exposed to the HPAI-H5N1 virus by allowing the flies to consume food containing the virus for 15 min, while the flies in group 1 were allowed to consume H5N1-free food and would serve as a negative control group. Group 2 flies were euthanatized immediately after H5N1 exposure, while group 3 were held at room temperature for 24 hr and euthanatized. The houseflies in the transmission of the HPAI-H5N1 virus were examined by challenging three groups of housefly homogenates into layer chickens via the oral drop. Morbidity and mortality were observed for 14 days, and virus shedding monitored via oropharyngeal swabs (OS) and cloacal swabs (CS), which were collected daily and determined by real-time reverse transcription-PCR and virus titration. Experimental challenge showed that all the chickens of groups 2 and 3 died within 7 days of inoculation. The OS had higher concentrations of virus than CS. Moreover, the chickens of group 2 had higher concentrations of virus shedding than the chickens of group 3. Immunohistochemistry detected the nucleoprotein of the type A influenza virus in all tissue samples collected, including the trachea, duodenum, pancreas, and brain. In summary, this study demonstrates that houseflies could serve as vectors in HPAI-H5N1 virus transmission in chickens under experimental conditions.

Molecular cloning and tissue distribution of the Toll receptor in the black tiger shrimp, Penaeus monodon.

The black tiger shrimp (Penaeus monodon) is economically important in many parts of the world, including Thailand. Shrimp immunity is similar to that of other invertebrate organisms; it consists of an innate immunity system. Toll or Toll-like receptors (TLRs) play an essential role in recognizing the cleaved form of the cytokine Spätzle, which is processed by a series of proteolytic cascades activated by secreted recognition molecules. We isolated a full-length Toll receptor from P. monodon. The cloned full-length sequence of the PmToll cDNA consists of 4144 nucleotides, containing a 5'-UTR with 366 nucleotides, a 3'-terminal UTR with 985 nucleotides, with a classical polyadenylation signal sequence AATAAA, a poly A-tail with 27 nucleotides, and an open reading frame coding for 931 amino acids. The deduced amino acid sequence of PmToll is a typical type I membrane domain protein, characteristic of TLR functional domains. It includes a putative signal peptide, an extracellular domain consisting of leucine-rich repeats, flanked by cysteine-rich motifs, a single-pass transmembrane portion, and a cytoplasmic TLR domain. PmToll was expressed in all tissues tested, including gill, hemocytes, heart, hepatopancreas, lymphoid organs, muscle, nerve, pleopod, stomach, testis, and ovary. The deduced amino acid of PmToll is closely related to that of other shrimp Tolls, especially FcToll. Further studies elucidating the mechanism of action of Tolls will be of benefit for understanding the defense mechanisms of this economically important aquatic species.

The potential of house flies to act as a vector of avian influenza subtype H5N1 under experimental conditions.

The objective of the present study was to determine the potential for house flies (Musca domestica L.) (Diptera: Muscidae) to harbour the avian influenza (AI) H5N1 virus. Laboratory-reared flies were experimentally fed with a mixture containing the AI virus. Exposed flies were washed with brain-heart infusion broth and followed by 70% alcohol before preparation of whole fly homogenate. The homogenate was inoculated into six 10-day-old embryonated chicken eggs (ECEs). Allantoic fluids were collected to determine the virus using the haemagglutination (HA) test, reverse transcription-polymerase chain reaction (RT-PCR) or quantitative real-time RT-PCR (RRT-PCR). In the first experiment, ECEs that were inoculated with the 50 AI virus exposed fly homogenates died within 48 h and HA and RT-PCR were positive for AI virus. In the second experiment, ECEs that were inoculated with only one fly died with positive HA test and RT-PCR. In the last experiment, a group of exposed flies was collected at 0, 6, 12, 24, 36, 48, 72 and 96 h post-exposure. Fly homogenates of each time point were tested by virus titration in ECEs and RRT-PCR. Virus titres declined in relation to exposure time. Furthermore, RRT-PCR results were positive at any time point. The present study shows that the flies may harbour the AI virus and could act as a mechanical vector of the AI virus.

Inhibition of Plasmodium falciparum proliferation in vitro by double-stranded RNA directed against malaria histone deacetylase.

Acetylation and deacetylation of histones play important roles in transcription regulation, cell cycle progression and development events. The steady state status of histone acetylation is controlled by a dynamic equilibrium between competing histone acetylase and deacetylase (HDAC). We have used long PfHDAC-1 double-stranded (ds)RNA to interfere with its cognate mRNA expression and determined the effect on malaria parasite growth and development. Chloroquine- and pyrimethamine-resistant Plasmodium falciparum K1 strain was exposed to 1-25 microg of dsRNA/ml of culture for 48 h and growth was determined by [3H]-hypoxanthine incorporation and microscopic examination. Parasite culture treated with 10 microg/ml pfHDAC-1 dsRNA exhibited 47% growth inhibition when compared with either untreated control or culture treated with an unrelated dsRNA. PfHDAC-1 dsRNA specifically blocked maturation of trophozoite to schizont stages and decreased PfHDAC-1 transcript 44% in treated trophozoites. These results indicate the potential of HDAC-1 as a target for development of novel antimalarials.

Expression of the catalase gene katA in starter culture Lactobacillus plantarum TISTR850 tolerates oxidative stress and reduces lipid oxidation in fermented meat product.

The catalase gene katA of Lactobacillus sakei SR911 was cloned and expressed in Escherichia coli UM2 and Lactobacillus plantarum TISTR850 under strong lactococcal promoter P59 in E. coli-lactococcus expression vector pIL1020. The L. plantarum TISTR850 is a catalase-deficient strain isolated from local fermented meat product. The recombinant L. plantarum TISTR850 was shown to decompose hydrogen peroxide, and catalase activity approximately three times higher that of natural catalase-producing strain L. sakei SR911. The recombinant protein was also detected by in situ activity staining of the catalase enzyme. The recombinant L. plantarum TISTR850 did not accumulate hydrogen peroxide under glucose-limited aerobic conditions and remained viable after 60 h of incubation. The recombinant and host strain L. plantarum TISTR850 were used as starter cultures in the fermented meat product, and lipid oxidation was monitored over a 7-day storage at 20 degrees C determined as thiobarbituric acid-reactive substances (TBARS) value. The lipid oxidation level in the fermented meat product seeded with the catalase genetically modified starter culture L. plantarum TISTR850 was significantly lower than that of the natural catalase-deficient strain.

Isolation of nisin-producing Lactococcus lactis WNC 20 strain from nham, a traditional Thai fermented sausage.

A total of 14,020 lactic acid bacteria (LAB) were isolated from nham and screened for bacteriocin production. One Lactococcus lactis strain WNC 20 produced a bacteriocin that not only inhibited closely related LAB, but also some food-borne pathogens including Listeria monocytogenes, Clostridium perfringens, Bacillus cereus and Staphylococcus aureus. Biochemical studies revealed that the bacteriocin was heat-stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10). The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K but not other proteases. The antimicrobial spectrum and some characteristics of this bacteriocin were nearly identical to that of nisin. The gene encoding this bacteriocin was amplified by polymerase chain reaction (PCR) with nisin gene-specific primer. Sequencing of this gene showed identical sequences to nisin Z as indicated by the substitution of asparagine residue instead of histidine at position 27. The ability of the bacteriocin produced by Lc. lactis WNC 20 may be useful in improving the food safety of the fermented product.

Detection of hepatopancreatic parvovirus (HPV) infection in Penaeus monodon using PCR-ELISA.

A rapid and sensitive PCR-ELISA has been developed for detection of hepatopancreatic parvovirus (HPV) in Penaeus monodon. The specific primer set amplified 156 bp fragment and could detect as a little as 0.01 fg of purified HPV DNA which equivalent to three viral particles. No cross-reactivity was observed when nucleic acid templates from white spot syndrome virus, yellow-head virus, monodon baculovirus and shrimp were tested. The crude DNA simple prepared from hepatopancreas can be used as DNA template and provide a favorable result. Using this technique for detection of HPV infection in 87 carrier shrimps revealed the higher sensitivity and efficiency of detection when compared to histological examination and conventional PCR. Sixty-two percent infection was detected by PCR-ELISA from samples with HPV negative diagnosed by histological examination. Therefore, this sensitive and specific method is promisingly useful for early detection of HPV infection in broodstock, carriers and for ex situ application where large numbers of samples can be analyzed simultaneously.

Transovarial transmission of sugarcane white leaf phytoplasma in the insect vector Matsumuratettix hiroglyphicus (Matsumura).

White leaf is a serious disease of sugarcane caused by phytoplasma. The disease is transmitted to the plant by the leafhopper Matsumuratettix hiroglyphicus (Matsumura). The reservoir of phytoplasma was suspected to be weeds that grow in sugarcane farming areas because they can be infected with phytoplasma and show symptoms similar to sugarcane white leaf. However in previous work we have demonstrated by RFLP and sequencing that this is not the case. Here we have reared M. hiroglyphicus through two generations by feeding them phytoplasma free sugarcane grown from tissue culture. By nested-PCR followed by sequencing, we demonstrated the presence of the phytoplasma in eggs, nymphs and adults of the first and second generations thereby showing transovarial transmission. We have also shown by in situ PCR that phytoplasmas were widely distributed throughout the body of the insect. RFLP and sequencing showed that the same phytoplasma was present in the vector and in the plant. Together, these data point to the leafhopper M. hiroglyphicus as the reservoir of phytoplasma that cause sugarcane white leaf disease.

Nucleotide sequence of a Thai isolate of Papaya ringspot virus type W.

The complete nucleotide sequence of a Thai isolate of Papaya ringspot virus (PRSV) type W (PRSV-W) was determined. The viral genome is 10,323 nucleotides (nts) long and contains an ORF encoding polyprotein 3,343 amino acids (aa) long, flanked with 5'- and 3'-non-coding regions (NCRs) of 85 and 206 nts, respectively. Out of the ten putative proteins P1 is the most variable (73.9% similarity) as compared to the PRSV type P (PRSV-P) sequences, while the CI protein is most conserved (99.1% similarity). The sequence similarity among the type W and P isolates also suggests that the P type arose from the W type. However, no significant difference between types P and W that would account for the host specificity was disclosed.

Recombinant Enterobacter amnigenus highly toxic to Anopheles dirus mosquito larvae.

The mosquito-larvicidal binary toxin of Bacillus sphaericus 2297 was expressed in Enterobacter amnigenus, a Gram-negative bacterium isolated from Anopheles dirus larvae gut. The toxin was placed under the regulation of various promoters in order to improve the expression level of the toxin. Amongst the recombinants obtained, E. amnigenus harboring pBS373, a plasmid which contains the toxin genes under the control of the native B. sphaericus promoter, expressed a significant amount of protein, comparable to that found in B. sphaericus 2297. In addition, this recombinant provided approximately twenty times higher toxicity against second-instar Anopheles dirus larvae when compared to B. sphaericus 2297. The procedure of obtaining this environmentally isolated bacterium from larvae gut and introducing the system for mosquito-larvicidal toxin synthesis is noteworthy. The promising result presented here provides a substantial degree of confidence for further field studies.

Determination of haemophilia A carrier status by mutation analysis.

A reliable method for determination of carrier status and genetic counselling is required for effective control of haemophilia. Linkage analysis is currently the most widely used method for this purpose; however, in cases where there is no prior family history and/or unavailability of informative polymorphic markers it is less applicable. Detection of a mutation characterized in each family may be an alternative method for determination of the carrier status. In this study, linkage analysis using four polymorphic DNA markers, and direct mutation analysis were compared to determine the carrier status in six unrelated Thai haemophilia A families, two with a family history and four without. In the two families with a family history of haemophilia A, the carrier and noncarrier statuses could readily be determined in eight females by either linkage or direct mutation analysis. In the four families without a family history, the polymorphic DNA markers for linkage analysis were informative in two families and uninformative in the other two. The carrier status could be excluded in all four female siblings of the patients in the former. However, the specific FVIII gene mutation was not observed in the mother of one patient, who should have carried the mutation. In the remaining two families with uninformative polymorphic DNA markers, the carrier and noncarrier statuses of four female members could only be determined by direct mutation analysis. Therefore, direct mutation analysis could circumvent the limitations of linkage analysis in the determination of haemophilia A carrier status in families without a previous history or informative polymorphic markers.

A rapid polymerase chain reaction based method for identification of the Anopheles dirus sibling species.

A simple polymerase chain reaction (PCR) based method was developed to differentiate the Anopheles dirus, species A, B, C and D in Thailand using specific primers designed from species specific sequences. The PCR protocol was optimized to obtain products of 120 bp, 75 bp, 60 bp and 172 bp for species A, B, C and D, respectively. This method used a cocktail of four primer sets to identify these An. dirus sibling species. The method is very sensitive as only a small portion of mosquito was required allowing the rest of the mosquito to be used for other analyses. Specimens also kept for up to 14 years could be analyzed unambiguously from either larvae or adult. This method is advantageous over other PCR-based methods for identification of malaria vectors because it does not require any specific DNA extraction. A mosquito specimen was homogenized in 1x PCR buffer, then the supernatant directly used for PCR identification, allowing a large number of samples to be processed at the same time. It provides a simple and rapid practical method for screening An. dirus species, which is essential in malaria vector epidemiological studies in Southeast Asia.

A non-stop, single-tube, semi-nested PCR technique for grading the severity of white spot syndrome virus infections in Penaeus monodon.

A single-tube, non-stop, semi-nested polymerase chain reaction (PCR) technique was developed for simultaneous detection and severity grading of white spot syndrome virus (WSSV) infections in the black tiger shrimp Penaeus monodon. The test uses 1 sense primer and 3 antisense primers that produce up to 3 PCR products (1100, 526 and 250 base pairs [bp]) depending upon the severity of infection. Specifically, heavy infections (> or = 2 x 10(4) viral particles) of WSSV produce all 3 fragments, while moderate infections (around 2 x 10(3) viral particles) produce 2 (526 and 250 bp) and light infections (20 to 200 viral particles) produce 1 (250 bp). In addition, the technique uses internal control primers that yield a shrimp characteristic fragment for non-infected samples and samples with a low quantity of viral target in order to assure integrity and reproducibility of the PCR assays. The non-stop, single-tube, semi-nested PCR technique is simple and convenient and can detect as little as 5 fg WSSV DNA (20 viral particles) in crude extracts of postlarval samples or extracts of pleopods and haemolymph from larger shrimp.

Isolation of bacterial strains colonizable in mosquito larval guts as novel host cells for mosquito control.

We screened for microorganisms that can be utilized as new host cells for mosquito larvicides. As long persistence in the environment is required of host cells, we examined the bacterial populations in the guts of mosquito larvae collected from natural breeding habitats. Larvae of Aedes aegypti and Culex quinquefasciatus were examined, and Bacillus species, particularly Bacillus cereus, were found to be the dominant bacterial species in their guts. To investigate the relationship between these Bacillus strains and the mosquito larvae, we re-introduced the bacteria into larvae of Aedes aegypti, C. quinquefasciatus and another common mosquito strain, Anopheles dirus. The cell numbers of Bacillus cereus strains Ae10 and Cx5 in the guts were consistent throughout a 7-d period without food supplementation, suggesting that these strains were able to colonize in the guts of the larvae. To confirm this, we introduced a plasmid containing a kanamycin resistance marker into Ae10 and Cx5 and fed these recombinant strains to C. quinquefasciatus larvae. Even when food was supplemented for 7 d, the recombinant strains, particularly Ae10, were still found in the guts. Under similar conditions, B. thuringiensis serovar israelensis c4Q2-72 was hardly detectable after 2 d, while Escherichia coli could not be detected at all. Their stable retention in mosquito larvae guts and the feasibility of genetic manipulation indicates these strains possess high potential as novel host cells for application in mosquito control.

Frameshift mutations with severe and moderate clinical phenotypes in Thai hemophilia A patients.

Six frameshift mutations in exon 14 of the factor VIII gene were identified in Thai hemophilia A patients. Although all these mutations created premature stop codons and expected to cause severe disease, the molecular defects and clinical severity were in discrepancy in some patients. Four mutations (delT3490, delACAC3618-21, delGA4429-30, and delA4658) were found in the patients with the severe clinical phenotype while two (delA3629-37 and insA4372-9) were observed in the patients who had moderate severity, with FVIII:C of 4.2 and 2.8%. The frameshift mutations in these two patients were due to deletion and insertion of an 'A' nucleotide in the stretches of 9As and 8As in codons 1191-4 and 1439-41, respectively. This indicates that deletion or insertion in the stretches of poly A nucleotides in exon 14 of the factor VIII gene is a likely cause of the moderate clinical severity in some cases of Thai hemophilia A patients.

Mutations of the factor VIII gene in thai hemophilia A patients.

Hemophilia A is a common X-linked bleeding disorder caused by mutations in the coagulation factor VIII gene. The entire coding and essential sequences of the factor VIII gene were generated by a combination of genomic DNA amplification and long reverse transcription-polymerase chain reaction (long RT-PCR) using factor VIII transcripts prepared from lymphocytes. Mutations were then screened by non-radioactive single strand conformation polymorphism (SSCP) analysis and characterized by DNA sequencing. We have identified six potentially pathogenic mutations in the factor VIII gene in Thai hemophilia A patients, including two nonsense mutations (R-5X and R1966X), three missense mutations (D542Y, G1850V, and G2325C), and a 4-bp insertion (ACTA) at codon 2245. Three of these mutations (D542Y, G2325C, and 4-bp insertion) have never been previously reported, and the ins2245 is the first example of such insertion probably causing factor VIII elongation. R1966X, D542Y, G1850V, and 4-bp insertion were associated with a severe hemophiliac phenotype whereas R-5X and G2325C were observed in moderately affected patients. Mutations in the factor VIII gene in Thai hemophilia A patients are likely to be heterogeneous. This study represents the first attempt to further the understanding of the molecular basis of hemophilia A in Thai.

Stable marker on flagellin gene sequences related to arabinose non-assimilating pathogenic Burkholderia pseudomallei.

Using PCR-based isolation and sequence analysis of the flagellin gene from two distinct biotypes of Burkholderia pseudomallei, a 15-bp deletion was found within the variable domain of the gene in isolates capable of assimilating arabinose (Ara+). This finding led to the development of a PCR-based method in order to differentiate and identify pathogenic B. pseudomallei for epidemiological study. A pair of specific primers was designed covering the 15-bp deletion region at the variable domain. PCR-amplification products of 176 and 191 bp in size were detected from 41 Ara+ isolates and 39 Ara - isolates of B. pseudomallei, respectively. Moreover, flagellin gene fragments of other bacterial species tested in this study were not amplified using these primers. The results suggest that the flagellin gene sequences of both B. pseudomallei biotypes in this region are stable and distinct. This method can be applied and useful for the epidemiological study of B. pseudomallei.

Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses.

Corresponding genomic regions of isolates of yellow head virus (YHV) from Thailand and gill-associated virus (GAV) from Australia were compared by RT-PCR and sequence analysis. PCR primers designed from sequences in the GAV ORF1b polyprotein gene amplified the corresponding 577 nucleotide region of the YHV genome. Comparison of the amplified region indicated 85.1% nucleotide and 95.8% amino acid sequence identity. YHV PCR primers designed to amplify a 135 nucleotide product previously described as a YHV diagnostic probe failed to amplify the corresponding product from GAV RNA. However, the cognate GAV sequence for this and another recently reported YHV sequence were located in an upstream region of the ORF1b gene. A comparison of these sequences indicated identities of 83.0 and 80.9% at the nucleotide level and 86.7 and 86.5% at the amino acid level, respectively. The data indicate that GAV and YHV are closely related but distinct viruses for which differential diagnostic probes can be applied.

PCR-RFLP analysis of the flagellin sequences for identification of Burkholderia pseudomallei and Burkholderia cepacia from clinical isolates.

The flagellin genes of four Burkholderia pseudomallei and two Burkholderia cepacia clinical isolates were studied by a polymerase chain reaction (PCR)-based isolation method using the same pair of primers. The PCR-amplification products of the isolates showed a single band of about 1.1 kb, which is similar to a type II B. cepacia flagellin reported previously. In order to distinguish these two species based on the flagellin PCR-amplified products, Pst I and Xho I restriction endonuclease analysis was performed. The results suggest that there is sufficient diversity within the flagellin sequences of the closely related Burkholderia species, B. pseudomallei and B. cepacia, to enable flagellin-type identification on the basis of the pattern of restriction fragments. In addition, the flagellin gene should be of considerable use as a genetic marker for clinical identification of these organisms.

Isolation and characterization of Synechococcus PCC7942 promoters: tRNApro gene functions as a promoter.

Promoter-active fragments of Synechococcus PCC7942 were isolated by transcriptional gene fusion to the promoterless beta-glucuronidase (GUS) gene of E. coli, which was used as a reporter gene. Several of the isolated promoter-active fragments expressed GUS activity in Synechococcus comparable to that of the lambdaPR promoter. Only 10% of the isolated promoter-active fragments also functioned in E. coli. The transcription initiation sites of the two promoter-active fragments, D13 and E3, were identified. The major transcription initiation sites of D13 and E3 in Synechococcus were located within the nucleotides TTTG and TTG respectively, which were identical to those corresponding to E. coli. The inferred -10 and -35 regions of D13 were TAAACT and TTGTAG respectively, which conformed to the E. coli sigma70 promoter. Immediately upstream of the E3 transcription initiation sites was the tRNApro (GGG) gene, which contained two regions exhibiting strong homology to the major promoter elements in eukaryotic tRNA genes, but did not contain the E. coli promoter element. Thus, the tRNApro gene can act as a promoter.