Induction of regulatory T cells by Opisthorchis viverrini.

Opisthorchis viverrini causes public health problems in South-East Asia. Recently, TGF-ß and IL-10 have been reported to increase in O. viverrini-infected hamsters but the sources of these cytokines are still unknown. In this study, the CD4+ T cells in infected hamsters were investigated. It was demonstrated that IL-4+ CD4+ T cells were significantly increased in hamster spleens and mesenteric lymph nodes (MLNs) during chronic infection. Interestingly, IL-10+ CD4+ T cells were also discovered at a significant level while Treg (T regulatory)-like TGF- ß+ CD4+ T cells were in MLNs of infected hamsters. Moreover, the CD4+ CD25+ Foxp3+ Treg cell response was significantly found both in spleens and MLNs in infected hamsters. The findings were then confirmed by development of T-cell clones against crude somatic antigens (CSAg) in immunized BALB/c mice. Five clones named TCC21, TCC23, TCC35, TCC41 and TCC108 were established. The TCC21 was found to be the TGF-ß+ CD4+ while TCC35, TCC41 and TCC108 were IL-4+ CD4+ and TCC23 was IFN-γ+ CD4+ . This TGF-ß+ CD4+ T clone showed an inhibitory function in vitro in mononuclear cell proliferation via TGF-ß-mediated mechanisms. This study indicated that O. viverrini-infected hamsters could induce TGF-ß+ CD4+ Treg-like cells. The CSAg-specific Tregs secreted high TGF-ß, and limited immune cell proliferation.

Identification of the Burkholderia pseudomallei bacteriophage ST79 lysis gene cassette.

AIMS: To identify and characterize the lysis gene cassette from the bacteriophage ST79 that lyses Burkholderia pseudomallei. METHODS AND RESULTS: Approximately 1·5 kb of ST79 lysis genes were identified from the phage genome data. It was composed of holin, peptidase M15A or endolysin, lysB and lysC. Each gene and its combinations were cloned into Escherichia coli and the lytic effects were measured. Co-expression of holin and peptidase M15A showed the highest lysis activity. Expression of holin, lysB/C or holin-peptidase M15A-lysB/lysC lysed the E. coli membrane, whereas peptidase M15A alone did not. The predicted transmembrane structures of holin and lysB/C indicated that they could be inserted into the bacterial membrane to form pores, affecting cell permeability and causing lysis. CONCLUSION: This is the first report of an investigation into the lysis genes of B. pseudomallei's lytic phage using E. coli as a model. SIGNIFICANCE AND IMPACT OF THE STUDY: Burkholderia pseudomallei, a Gram-negative bacterium causing an infectious disease, is intrinsically resistant to several antibiotics, and a vaccine is not available. The lysis genes of ST79, the first reported lytic bacteriophage of B. pseudomallei, were characterized. The development of ST79 as an alternative treatment for skin ulceration, for example, or to be used as a gene cloning tool for B. pseudomallei may be possible with this knowledge.

Use of a low-dose steroid as an adjunct in the treatment, in mice, of severe sepsis caused by Burkholderia pseudomallei.

Human melioidosis caused by Burkholderia pseudomallei is a severe septic disease that is associated with high mortality, even under appropriate antibiotic treatment. The therapeutic effects of low-dose hydrocortisone plus ceftazidime, and of ceftazidime alone, have recently been investigated in the treatment of acute, severe sepsis caused by B. pseudomallei, both in normal BALB/c mice and in BALB/c mice with streptozotocin-induced diabetes. The mice were infected and then treated intravenously, from day 1 or day 2 post-infection, with saline (as a control, given twice daily for 10 days), low-dose hydrocortisone (given in twice-daily doses of 5 mg/kg, for 5 days) plus ceftazidime (given in twice-daily doses of 1200 mg/kg, for 10 days), or the same doses of ceftazidime alone. Although the infected, untreated mice all died within 14 days, almost all of the treated animals were still alive at the end of the follow-up, 30 days post-infection. The addition of the steroid appeared to have no benefit, with bacterial loads and plasma concentrations of tumour necrosis factor, aspartate aminotransferase, alanine aminotransferase and creatinine decreasing similarly in all the treated groups. The infected diabetic mice given hydrocortisone-ceftazidime from day 1 (but not those given just ceftazidime from day 1) showed an increase in their blood glucose concentrations. When infected mice were treated with the low-dose steroid and lower doses of the antibiotic (in twice-daily doses of 120-600 mg/kg), the steroid not only offered no apparent benefit but seemed to reduce survival. It therefore appears that low-dose hydrocortisone, as an adjunct to antibiotic treatment, does not provide benefit in the treatment of murine melioidosis and may have negative effects on human cases of the disease who have diabetes mellitus.

Isolation of fetal nucleated red blood cell from maternal blood using immunomagnetic beads for prenatal diagnosis.

The immunomagnetic beads method for isolation of fetal nucleated red blood cells (FNRBCs) from peripheral blood of 78 pregnant women for prenatal diagnosis was developed. The study subjects were classified into 8-10 and 11-14 weeks of gestation (n = 39 each). Peripheral blood cells were divided into two for the FNRBCs isolation using two protocols, one with anti-CD45 depletion followed by anti-CD71 and anti-GPA monoclonal antibodies and another without CD45 depletion. The use of CD45 depletion gave a slightly higher number of sorted cells but not significantly different (p > 0.05). The percentage of CD71+ and GPA+ cells obtained from 8-10 weeks and 11-14 weeks of gestation was not different (p > 0.05). The sensitivity in determining the sorted FNRBCs for male fetal sex by PCR using 8-10 and 11-14 weeks of gestation was generally 50 and 69%, respectively. The method so developed is simple and cost effective and may thus be applied for prenatal diagnosis.

Role of RelA and SpoT in Burkholderia pseudomallei survival, biofilm formation and ceftazidime tolerance during nutritional stress.

Burkholderia pseudomallei a saprophyte found in soil and stagnant water is the causative agent of human melioidosis, an often cause fatal disease. B. pseudomallei is intrinsically resistant to many antibiotics. The stringent response is a global bacterial adaptation process in response to nutritional limitation and is mediated by the alarmone (p)ppGpp, which is produced by two proteins, RelA and SpoT. In order to test whether the stringent response is involved in ceftazidime tolerance, biofilm formation, and bacterial survival in the soil microcosm, B. pseudomallei strain K96243 and its isogenic ΔrelA and ΔrelAΔspoT mutants were grown in rich and nutrient-limited media. In nutrient-limiting conditions, both the wild type and mutants were found to be up to 64-times more tolerant to ceftazidime than when grown in rich culture conditions. Moreover, the biofilm formation of all bacterial isolates tested were significantly higher under nutrient-limiting conditions than under nutrient-rich conditions. The ΔrelAΔspoT mutant produced less biofilm than its wild type or ΔrelA mutant under nutrient-limiting conditions. The survival of the ΔrelAΔspoT double mutant cultured in 1% moisture content soil was significantly decreased compared to the wild type and the ΔrelA mutant. Therefore, the RelA/SpoT protein family might represent a promising target for the development of novel antimicrobial agents to combat B. pseudomallei.

Retrospective Study on Fatal Melioidosis in Captive Zoo Animals in Thailand.

Melioidosis is caused by Burkholderia pseudomallei and is an important zoonotic infectious disease causing high mortality from fulminant septicaemia in humans and a wide variety of animal species. The incidence of fatal melioidosis in zoo animals has been significant in many Thai zoos. A total number of 32 cases were evaluated throughout the Thai zoo animal populations. The highest prevalence of disease has been reported from the north-eastern region followed by the zoos in the southern part of the country, approximately 47% and 38%, respectively, while the other zoos reported sporadic infections. Herbivores and non-human primates were the most commonly affected animals with incidences of 59% and 28%, respectively. This appears to be a seasonal correlation with the highest incidence of melioidosis in zoo animals reported in the rainy season (44%) or subdivided monthly in June (19%) followed by September and November (16% and 12%, respectively). The route of infection and the incubation period still remain unclear. This retrospective study examined the clinical presentation in various zoo species, pathological findings and epidemiological data as well as conducting an in depth literature review.

Drug susceptibility and biofilm formation of Burkholderia pseudomallei in nutrient-limited condition.

Burkholderia pseudomallei is the causative agent of melioidosis, which can form biofilms and microcolonies in vivo and in vitro. One of the hallmark characteristics of the biofilm-forming bacteria is that they can be up to 1,000 times more resistant to antibiotics than their free-living counterpart. Bacteria also become highly tolerant to antibiotics when nutrients are limited. One of the most important causes of starvation induced tolerance in vivo is biofilm growth. However, the effect of nutritional stress on biofilm formation and drug tolerance of B. pseudomallei has never been reported. Therefore, this study aims to determine the effect of nutrient-limited and enriched conditions on drug susceptibility of B. pseudomallei in both planktonic and biofilm forms in vitro using broth microdilution method and Calgary biofilm device, respectively. The biofilm formation of B. pseudomallei in nutrient-limited and enriched conditions was also evaluated by a modified microtiter-plate test. Six isolates of ceftazidime (CAZ)-susceptible and four isolates of CAZ-resistant B. pseudomallei were used. The results showed that the minimum bactericidal concentrations of CAZ against B. pseudomallei in nutrient-limited condition were higher than those in enriched condition. The drug susceptibilities of B. pseudomallei biofilm in both enriched and nutrient-limited conditions were more tolerant than those of planktonic cells. Moreover, the quantification of biofilm formation by B. pseudomallei in nutrient-limited condition was significantly higher than that in enriched condition. These data indicate that nutrient-limited condition could induce biofilm formation and drug tolerance of B. pseudomallei.

Comparison of the polymerase chain reaction and serologic tests for diagnosis of septicemic melioidosis.

For diagnosis of melioidosis, we compared polymerase chain reaction (PCR)-based DNA detection and three serologic methods with the culture method currently used as gold standard. The diagnostic values of the serologic methods were evaluated in 130 patients. All these patients resided in an endemic area. An enzyme-linked immunosorbent assay (ELISA) gave slightly higher specificity (86.2%) than a dot immunoassay (DOT) (85.3%), but was superior to an indirect hemagglutination assay (IHA) (79.8%). The sensitivities of the DOT (85.7%) and ELISA (71.4%) were considerably higher than that of IHA (61.9%). However, the PCR was the most sensitive (95.2%) and specific (91.7%). Nevertheless, DOT and ELISA are more practical for local hospitals. With the high negative predictive value of both the ELISA (94.0%) and DOT (96.9%) in a high prevalence area, clearly these methods can rule out most of the non-melioidosis patients.

Use of multiplex PCR patterns as genetic markers for Burkholderia pseudomallei.

A simple PCR-based typing method was developed to differentiate between strains of Burkholderia pseudomallei. Two pairs of primers, based on sequences from two specific DNA probes, were used to amplify the bacterial DNA by multiplex PCR. We evaluated the PCR method for epidemiological typing of B. pseudomallei and compared this with restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) methods. In RFLP, the DNA of B. pseudomallei was digested with HindIII and the pKKU-S23L was used as a probe while 5' GTTTCGCTCC 3' primer was used in RAPD. DNA was obtained from 37 B. pseudomallei environmental and clinical isolates from humans or animals. These isolates were also classified by their ability to assimilate L-arabinose. A total of 21 type patterns were identified by multiplex PCR. Among human and animal isolates, multiplex PCR revealed ten types, all of which were arabinose negative (Ara-), whereas six of the 11 types of environmental isolates were Ara-. There are two environmental patterns that also were found in clinical isolates. The RFLP technique showed 12 different types in the 37 isolates, and three of these contained both Ara+ and Ara- isolates. The RAPD technique revealed 33 different types in the 37 isolates. Multiplex PCR, therefore, is the genetic marker that best correlates with the ability of the organism to assimilate L-arabinose. Moreover, two types (M4, M15) correlated with disseminated septicemic melioidosis in the northeast Thailand. If a greater number of isolates are tested, the multiplex PCR technique may prove to be useful for rapid epidemiological typing of B. pseudomallei.

Immunology and molecular biology of Opisthorchis viverrini infection.

Opisthorchiasis is the major public health problems in Laos PDR and Thailand. The disease becomes chronic and persists for many years, leading to hepatobiliary disease and cholangiocarcinoma. Less severe manifestations include cholangitis, chronic cholecystitis and cholelithiasis. A significant degree of humoral and cell mediated immune responses to the parasite can be detected both in patients and animal models. The patients IgG levels appear to correlate with gall bladder size and dysfunction and correlated significantly with opisthorchis egg count and decrease after treatment. However, the possible significance of these immune responses to protective immunity is presently unknown. The development of immunodiagnostic method for Opisthorchis viverrini detection has been attempted. The components with molecular weight >116, 89, 78 and 20 kDa appear to be specifically associated with the somatic extract of adult fluke. The 89 kDa protein is the most prominent component found in the in vitro culture fluid of adult worms and the metacercarial extract that can be a candidate with significant immunodiagnostic potential. Highly specific and sensitive monoclonal antibodies for O. viverrini antigens were prepared to detect parasite antigens in stool and antibody in serum. Information regarding the molecular approaches of O. viverrini is very limited. The genome of O. viverrini has neither CpG nor A methylated as found in other parasites. The total length O. viverrini ribosomal DNA is approximately 13 kb. and the presence of a highly repeated DNA specific for the parasite was demonstrated. A O. viverrini specific DNA probe was constructed and PCR based detection with high specificity for amplification of the repeated sequences is performed to detect the presence of eggs' DNA in stool samples in comparison with classical methods.

Comparison of three PCR primer sets for diagnosis of septicemic melioidosis.

Several sets of PCR primers have recently been developed for detection of Burkholderia pseudomallei. In this report, the performance of 16S rRNA gene primers (16S), rRNA spacer gene primers (spacer), and 'LPS' primers (LPS) were compared. All primer sets were tested by PCR amplification of the same DNA samples extracted from blood specimens of 46 patients from northeastern Thailand, of which 29 had melioidosis based on blood culture as a gold standard. The sensitivities were 41, 35.7, and 31% while the specificities were 47, 59, and 100% for the 16S, spacer, and LPS primers, respectively. The positive predictive values were 60, 59, and 100%, while negative predictive values were 35, 34, and 46%, for these primers. The low sensitivity of PCR was suspected to be because of small numbers of bacteria in the samples. In addition, one primer set could not detect all B. pseudomallei strains. To make PCR for melioidosis more practical, bacterial concentration steps must be added. Lastly, mixed infection of patients in endemic areas may be the cause of controversial false positive PCR results, and should be further investigated.

Ribotyping of Burkholderia pseudomallei from clinical and soil isolates in Thailand.

Burkholderia pseudomallei is a soil saprophyte that causes melioidosis in humans and animals. Restriction fragment length polymorphism of the ribosomal DNA regions (ribotyping) were analyzed in 577 isolates comprising 371 clinical and 206 soil isolates collected throughout Thailand in 1997. A total of 77 distinct ribotype patterns consisting of 2-9 bands with sizes ranging from 2.8 to >23 kb were found. Twelve major ribotypes were identified of which types 3, 8 and 23 were commonly found (278/577, 48.2%) in both clinical (217/371, 58.5%) and environmental isolates (61/206, 29.6%). Three unique environmental types were found whereas a unique clinical type was not observed. Even though ribotypes show high heterogeneity in the rDNA region, the unique environmental patterns were clearly different from the clinical patterns as clearly seen by UPGMA dendrogram. Moreover, the three major types (types 3, 8 and 23) were discovered in nearly half of B. pseudomallei isolates. Subtyping of these major ribotypes in correlation with clinical profiles may help researchers to identify the virulence factor of the organism.

Use of culture-filtrated antigen in an ELISA and a dot immunoassay for the diagnosis of melioidosis.

An enzyme-linked immunosorbent assay (ELISA) and a dot immunoassay with culture-filtrated antigen were developed for detection of Burkholderia pseudomallei specific antibodies in melioidosis patients. Sixty-eight sera of bacteriologically confirmed melioidosis patients, 45 sera of other bacterial infected patients and 80 sera of healthy blood donors from endemic area were investigated. The samples were subjected to those assays im comparison with indirect hemagglutination (IHA). The sensitivity, specificity, positive and negative predictive values in this dot immunoassay were 94.1%, 99.2%, 98.5% and 96.9%, respectively, with cut-off dilution at 1:4,000, whereas those in ELISA were 92.6%, 96.8%, 94.0% and 96.0%, respectively, with cut-off value of OD = 0.47 at 490 nm. Meanwhile, those in IHA were 64.7%, 93.6%, 84.6%, 83.0% respectively, with a cut-off value of > or = 1:80. The results in this study demonstrated that the dot immunoassay was more reliable and rapid than ELISA as the serological test for diagnosis of melioidosis.

Characterization of Pseudomonas pseudomallei antigens by SDS-polyacrylamide gel electrophoresis and western blot.

Immunological characterization of various Pseudomonas pseudomallei preparations was carried out by SDS-PAGE and Western blot using sera from infected humans and from patients with other bacterial infections. Somatic (SOM) and partially purified cell extracts (PCE) gave more complex SDS-PAGE patterns: M(r) ranged from 86 to 12.7 and 48 to 10 kDa, respectively. The culture-filtrated antigens (CF) from 3 different kinds of synthetic media consisted of fairly simple profiles with common bands M(r) of 40, 26 and 16 kDa. PCE and CF reacted specifically with infected human sera; SOM did not. The components with M(r) of 40 kDa in CF reacted consistently with all infected sera but failed to react with sera infected with Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, Proteus mirabilis, Salmonella spp., Staphylococcus aureus, Streptococcus spp., Pseudomonas aeruginosa and P. stutzeri. This peptide was demonstrated to be a major component in CF thus suggesting its potential for development of immunodiagnostic methods for melioidosis.

Retrospective study on the diagnostic value of IgG ELISA, dot immunoassay and indirect hemagglutination in septicemic melioidosis.

Three serological methods for diagnosis of melioidosis were compared with the culture method currently used as the "gold standard". The diagnostic values of the serological methods were evaluated retrospectively in 306 patients residing in an endemic area. The enzyme-linked immunosorbent assay (ELISA), using affinity purified antigen for detecting specific IgG antibody, showed a slightly higher specificity (86.0%) than the dot immunoassay (DOT) (84.0%) and both were superior to indirect hemagglutination (IHA) (72.0%). The sensitivity of DOT (96.4%) and ELISA (85.7%) were considerably higher than that of IHA (50.0%). The primary benefit of the high negative predictive value of both ELISA (96.4%) and DOT (99.0%) in an area of high prevalence is the ability to rule out most of the non-melioidosis patients.

Cytokine expression in hamsters experimentally infected with Opisthorchis viverrini.

The cytokine mRNA expression of IL-12, IFN-gamma, TGF-beta, IL-4, and IL-10 were investigated in spleen, liver and mesenteric lymph nodes (MLN) in hamsters experimentally infected with Opisthorchis viverrini. Animals were infected with 5, 25 or 100 metacercariae (Mc) and examined by RT-PCR and real-time PCR at 2 weeks, 2 and 6 months after infection. The cytokine expression was compared using HPRT. The IL-12 was significantly expressed at 2 weeks in the liver of the 5- and 25-Mc-infected groups. It is correlated with the inflammation intensity found in the liver at the same time. The production of IFN-gamma was not increased. The significant increase in expression of IL-10 was observed in the 6-month group in the spleen, which may suppress the Th1 and lead to a Th2 response. The IL-4 and TGF-beta expressions in MLN were significantly increased, and correlated with the dose of infection, especially in the 6-month groups. The TGF-beta level in MLN was 15-fold higher than in the uninfected control, compared to a twofold increase in spleen and liver. Because this parasite resides in the bile duct, the regulatory cytokine levels of mucosal immunity were enhanced more than those in systemic immunity. These results indicate the predominance of Th2 responses in chronic O. viverrini infection, and the high level of TGF-beta may inhibit the immune functions, which allows the parasites to evade host immune response.

Detection of Burkholderia pseudomallei in blood samples using polymerase chain reaction.

A highly sensitive, specific, rapid and simple method to detect Burkholderia pseudomallei in blood samples was developed. Two 22-base oligonucleotide primers, based on sequences from a specific DNA probe, were used for amplification of bacterial DNA by the polymerase chain reaction (PCR). Amplification with these primers yielded a 178-base pair product in 100 clinical isolates of B. pseudomallei. As little as 0.5 fg of B. pseudomallei DNA was detectable by this method. Experiments involving inoculation of the organism into uninfected blood samples showed that the method could be used to detect as few as 1 bacterial cell ml-1 of whole blood. Non-specific amplification of other bacterial DNAs from 18 samples of bacteria was not observed. Blood samples from seven patients proven to have melioidosis by haemoculture were positive using these primers. The total time required for sample processing, amplification and visualization was approximately 3.5 h. The high sensitivity, rapidity and simplicity of this method should make it valuable for diagnosis, monitoring of drug treatment and for epidemiological studies of the melioidosis.

Construction of a specific DNA probe for diagnosis of melioidosis and use as an epidemiological marker of Pseudomonas pseudomallei.

Pseudomonas pseudomallei is a causative agent of melioidosis. The disease manifestations range from fulminant sepsis to asymptomatic seroconversion. In septicemic cases, a mortality rate of 80-90% is reported. Rapid and specific diagnosis has become important to the clinical microbiology laboratory. We have developed a P. pseudomallei-specific DNA probe. The cloned fragment, herein designated pKKU-S23L, contained 1.5 kb of P. pseudomallei chromosomal DNA. A radioactively labelled pKKU-S23L insert could detect 1.5 ng of its genomic DNA or 40,000 P. pseudomallei cells. The probe was highly specific for P. pseudomallei DNA and did not cross-hybridize with DNAs prepared from other related bacteria. Using pKKU-S23L as a probe in total cellular DNA digestions and Southern blot hybridization, we were able to classify 60 P. pseudomallei clinical isolates obtained from individual melioidosis patients into eight categories. Therefore, this probe has a potential not only for use in development of specific detection of bacterial DNA in clinical specimens but also for application in epidemiological studies of P. pseudomallei.

Development of a PCR-based method for the detection of Opisthorchis viverrini in experimentally infected hamsters.

Opisthorchis viverrini infection is an endemic disease that causes a serious public health problem in southeast Asia, especially in northeast Thailand. We have developed a PCR method using a pair of primers named OV-6F/OV-6R for detecting O. viverrini eggs in stool samples and compared it with Stoll's egg-count method. The primers were designed based on the pOV-A6 specific DNA probe sequence which gave a 330 base pair product. The PCR method can detect a single egg in artificially inoculated faeces or as little as 2 x 10(-17) ng of O. viverrini genomic DNA. The method gave 100% sensitivity in all hamster groups except in animals exposed to the lowest intensity of infection (1 metacercaria/hamster). In the first month of infection, the PCR method was more sensitive than using the egg-count method in all infected groups especially in the light infections. The PCR method was also successfully used in monitoring a therapeutic study. Since the PCR method showed no cross-reaction with Heterophyid flukes, it can be useful for specific identification of O. viverrini eggs in stool samples without the risk of false positives. It also has great potential for application in clinical epidemiological studies.