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Background & Objectives: El Tor Vibrio cholerae O1 carrying ctxB C trait, so-called El Tor variant that causes more severe symptoms than the prototype El Tor strain, first detected in Bangladesh was later shown to have emerged in India in 1992. Subsequently, similar V. cholerae strains were isolated in other countries in Asia and Africa. Thus, it was of interest to investigate the characteristics of V. cholerae O1 strains isolated chronologically (from 1986 to 2009) in Thailand., Methods: A total of 330 V. cholerae O1 Thailand strains from hospitalized patients with cholera isolated during 1986 to 2009 were subjected to conventional biotyping i.e., susceptibility to polymyxin B, chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test. The presence of ctxA, ctxB, zot, ace, toxR, tcpA C , tcpA E, hlyA C and hlyA E were examined by PCR. Mismatch amplification mutation assay (MAMA) - and conventional- PCRs were used for differentiating ctxB and rstR alleles., Results: All 330 strains carried the El Tor virulence gene signature. Among these, 266 strains were typical El Tor (resistant to 50 units of polymyxin B and positive for CCA and VP test) while 64 had mixed classical and El Tor phenotypes (hybrid biotype). Combined MAMA-PCR and the conventional biotyping methods revealed that 36 strains of 1986-1992 were either typical El Tor, hybrid, El Tor variant or unclassified biotype. The hybrid strains were present during 1986-2004. El Tor variant strains were found in 1992, the same year when the typical El Tor strains disappeared. All 294 strains of 1993-2009 carried ctxBC ; 237 were El Tor variant and 57 were hybrid., Interpretation & Conclusions: In Thailand, hybrid V. cholerae O1 (mixed biotypes), was found since 1986. Circulating strains, however, are predominantly El Tor variant (El Tor biotype with ctxB C).

Episodes of cholera stemming from indigenous Vibrio cholerae strains in Australia are mainly associated with environmental sources. In the present study, 10 V. cholerae O1 strains of Australian origin were characterized. All of the strains were serogroup O1 and their conventional phenotypic traits categorized them as belonging to the El Tor biotype. Genetic screening of 12 genomic regions that are associated with virulence in V. cholerae showed variable results. Analysis of the ctxAB gene showed that the Australian environmental reservoir contains both toxigenic and non-toxigenic V. cholerae strains. DNA sequencing revealed that all of the toxigenic V. cholerae strains examined were of ctxB genotype 2. Whole genome PFGE analysis revealed that the environmental toxigenic V. cholerae O1 strains were more diverse than the non-toxigenic environmental O1 strains, and the absence of genes that make up the Vibrio seventh pandemic island-I and -II in all of the strains indicates their pre-seventh pandemic ancestry.

The aim of the present study was to genotypically characterize Vibrio cholerae strains isolated from cholera patients in various provinces of Thailand. Two hundred and forty V. cholerae O1 strains, isolated from patients with cholera during two outbreaks, i.e. March 1999-April 2000 and December 2001-February 2002, in Thailand, were genotypically characterized by NotI digestion and pulsed-field gel electrophoresis (PFGE). In total, 17 PFGE banding patterns were found and grouped into four Dice-coefficient clusters (PF-I to PF-IV). The patterns of V. cholerae O1, El Tor reference strains from Australia, Peru, Romania, and the United States were different from the patterns of reference isolates from Asian countries, such as Bangladesh, India, and Thailand, indicating a close genetic relationship or clonal origin of the isolates in the same geographical region. The Asian reference strains, regardless of their biotypes and serogroups (classical O1, El Tor O1, O139, or O151), showed a genetic resemblance, but had different patterns from the strains collected during the two outbreaks in Thailand. Of 200 Ogawa strains collected during the first outbreak in Thailand, two patterns (clones)--PF-I and PF-II--predominated, while other isolates caused sporadic cases and were grouped together as pattern PF-III. PF-II also predominated during the second outbreak, but none of the 40 isolates (39 Inaba and 1 Ogawa) of the second outbreak had the pattern PF-I; a minority showed a new pattern--PF-IV, and others caused single cases, but were not groupable. In summary, this study documented the sustained appearance of the pathogenic V. cholerae O1 clone PF-II, the disappearance of clones PF-I and PF-III, and the emergence of new pathogenic clones during the two outbreaks of cholera. Data of the study on molecular characteristics of indigenous V. cholerae clinical isolates have public-health implications, not only for epidemic tracing of existing strains but also for the recognition of strains with new genotypes that may emerge in the future.

Objective: To determine virulence associated-genes and ribotypes of Vibrio cholerae epidemic strains isolated from cholera patients in Thailand., Method: A total of 240 V. cholerae El Tor, O1 strains, isolated from patients with cholera in Thailand during two different periods, i.e. 1999-2000 (200 strains; 193 Ogawa and 7 Inaba) and 2001-2002 (40 strains; all Inaba), were analyzed for the presence of virulence genes, namely ctxA, ctxB, zot, ace, toxR, tcpA, hlyA, nanH and ninT by PCR. For ribotyping, genomic DNA segments of the 240 strains and 10 reference V. cholerae strains isolated before 1999 from Thailand and elsewhere were digested with BglI endonuclease, subjected to a 0.8% agarose gel electrophoresis, blotted onto a nylon membrane and probed with enzyme-labeled Escherichia coli rRNA. The DNA bands were visualized by autoradiography., Results: Genes encoding the A and B subunits of CT, Zot, Ace, ToxR, TcpA, HlyA, NanH and NinT could be amplified from all of the 10 V. cholerae O1 reference strains and from 239 of the 240 studied isolates. One Inaba isolate of 2001-2002 gave only amplicons of toxR and hlyA. For ribotyping, the 10 reference strains revealed six different patterns designated A to F. None of the 240 strains isolated in Thailand during the two periods had the A-C, E and F ribotypes. The isolates of 1999-2000 revealed ribotype D and three other ribotypes, designated G, H and I. The majority of the isolates of 2001-2002 showed ribotype G. The remaining showed other new ribotypes, J and K., Conclusions: The clinical V. cholerae isolates of two epidemics from Thailand showed a sustained appearance of one epidemic V. cholerae clone, and a constant, but gradual and minor change in the genetic constituent of the other V. cholerae strains as indicated by the change of the ribotypes of the strains in the two study periods. Moreover, we found that a V. cholerae strain which cannot produce CT, Zot, Ace, TcpA, NanH and NinT can still cause symptomatic cholera.

An oral cholera vaccine made up of three Vibrio cholerae antigens, i.e. lipopolysaccharide (LPS), recombinant toxin co-regulated pili (rTcpA) and heat-treated cholera toxin (H-CT) has been developed in six different formulations. Eight-week-old Wistar rats were divided into nine groups and immunized as follows: the first group received the oral vaccine 1 consisting of the three antigens (LPS, rTcpA and H-CT) associated with a liposome (L) and bacterial CpG-DNA (ODN#1826). The rats of groups 2 and 3 received oral vaccines 2 and 3 consisting of the liposome-associated three antigens with and without non-bacterial CpG-DNA (ODN#1982), respectively. Rats of groups 4 received oral vaccine 4 consisting of the three antigens mixed with the ODN#1826, similar to vaccine 1, but without liposome. Rats of groups 5 and 6 received oral vaccines 5 and 6 consisting of the three antigens with and without ODN#1982, respectively, similar to vaccines 2 and 3, but without liposome. Rats of groups 7, 8 and 9 received oral placebos, namely liposomes (L), ODN#1826 (CpG), and vaccine diluent, i.e. 5% NaHCO3 solution, respectively. All vaccines were given in three doses at 14-day intervals. It was found that the combination of liposome and ODN#1826 in vaccine 1 evoked the highest immune response to V. cholerae antigen compared to other vaccine formulations and placebos, as measured by the appearance of antigen-specific antibody-producing cells in the intestinal lamina propria. The immunogenicity according to the magnitude of the immune response was: V1>V2=V3>V4>V5=V6>V7=V8=V9. The results of this study indicate that CpG-DNA and liposome are effective mucosal adjuvants for an oral cholera vaccine prepared from refined V. cholerae antigens and their combination seems to be synergistic. The potential role of liposome as a vaccine delivery vehicle has been confirmed.

Hybridomas secreting specific monoclonal antibodies (MAbs) to Vibrio cholerae serogroup O139 were produced. Six monoclones (hybridomas) secreting MAbs specific only to lipopolysaccharide of V. cholerae O139 strains and which did not cross-react to 137 strains of other enteric microorganisms were obtained. These clones were designated 12F5-G11, 12F5-G2, 15F5-H5, 5B9-F8, 14C9-D2, and 6D2-D8. The immunoglobulin (Ig) heavy chain isotypes secreted by these clones were IgG2b, IgG2b, IgG2b, IgM, IgG2b, and IgG3, respectively. Clone 12F5-G11 was selected for mass production of MAb, which was used as a detection reagent in the antigen detection assay for diagnosis of cholera caused by V. cholerae O139, and this assay was compared to the conventional bacterial isolation method. Five batches of rectal swab cultures in alkaline-peptone water were collected from 6,497 patients with watery diarrhea. These were 6,310 patients admitted to Bamrasnaradura Infectious Diseases Hospital, 16 patients from Krung Thon Hospital, 78 patients from Bangkok Children's Hospital, 19 patients from Karen refugee camps, and 74 Indian patients from the National Institute of Cholera and Enteric Diseases, Calcutta, India. The V. cholerae O139 isolations from the rectal swab cultures and the antigen detection assays (i.e., the MAb-based dot-blot ELISA) were performed by different persons of different laboratories, and the results were revealed after all specimens had been tested. Of the 6,497 samples tested, the dot-blot ELISA correctly identified 42 of 42 V. cholerae O139-positive samples and gave a result of positive for three samples which were culture negative for V. cholerae O139. The diagnostic sensitivity, specificity, and efficacy of the dot-blot ELISA were 100, 99.95, and 99.26%, respectively. The ELISA is easy to perform and relatively inexpensive. It can test multiple samples at a single time, does not require special equipment, and does not produce great quantities of contaminated waste. Most of all, it reduces the diagnostic time from at least 2 days for the bacterial isolation to less than 90 min. The assay is recommended as a rapid screening test of cholera cases caused by V. cholerae O139.

Three formulations of oral cholera vaccine were compared with respect to their immunogenicity and protective ability in a rat ileal loop model. Eight-week-old Wistar rats were divided into five groups. The first group received orally vaccine A consisting of liposome-associated V. cholera lipopolysaccharide, fimbriae and procholeragenoid, whereas the rats of groups 2 and 3 received orally vaccines B and C consisting of heatkilled fimbriated and non-fimbriated whole cell V. cholerae, respectively. Rats of groups 4 and 5 were controls that received orally liposomes alone and normal saline solution, respectively. It was found that vaccine A elicited stronger immune responses to all three V. cholerae antigens. The antibody responses were detected in both serum and intestinal lavage samples. Vaccine B elicited only modest serum and intestinal responses to V. cholerae fimbriae (anti-F). No detectable immune response was found in rats of group 3 immunized with vaccine C. Rats immunized with vaccines A and B had a similar order of magnitude of numbers of vibrios adhered to their intestinal mucosa. These numbers were less than those associated with the intestinal tissues of control rats of groups 4 and 5 by about two orders of magnitude. Although without any detectable immune response, rats of group 3 that were immunized with vaccine C showed some reduction in numbers of vibrios associated with their intestinal mucosa. The numbers of vibrios recovered from the intestinal segments of rats of all treatment groups were in the order group 1 = 2 < 4 = 5. Electron micrography also revealed patches of vibrio colonization on the mucosa of rats of groups 3, 4 and 5. These features were not found in the groups vaccinated with vaccines A and B. The inhibition of vibrio colonization afforded by the vaccines was biotype- and serotype-non-specific. The results suggest that the heat-killed whole cell fimbriated V. cholerae may be an alternative vaccine preparation to the liposome-associated refined antigen vaccine at a lower cost.

A "cholera diagnostic kit" was developed for sensitive, specific, rapid, and inexpensive detection of Vibrio cholerae 01. The monoclonal antibody specific to antigen A of Vibrio cholerae 01 was used as an antigen detection reagent and the principle of dot-blot ELISA was adopted. The kits were used in seven Regional Medical Sciences Centres, Ministry of Public Health, located at various regions of Thailand where diarrhea occurs frequently. Diagnostic efficiency of the kits in the detection of Vibrio cholerae 01 from rectal swabs of the diarrheic patients and their household contacts was evaluated in comparison with the conventional culture method. The two methods were found to have excellent degree of agreement (kappa values > 95%). The dot-blot ELISA has several advantages over the culture methods, ie rapid (dot-blot ELISA takes 1-2 hours while the culture method takes at least two days) and inexpensive. It requires no sophisticated equipment. The procedure is not complicated thus it is easy to train personnel. The diagnostic kits are recommended for use in the detection of severe diarrhea caused by V. cholerae 01 not only in hospitals and health centres where adequate treatment of the patients is required as a life-saving measure but also for early recognition of cholera cases and their contacts so that other action, ie prevention and control of outbreaks and surveillance can be promptly implemented.

Colonization of V. cholerae O1 in vivo is known to be a non-invasive type which the vibrios are confined only to the intestinal tissues. The pathway by which the vibrio antigens reach the lymphoid cells and subsequently give rise to the immune responses is not entirely clear. Thus, experiments were performed in experimental rats by inoculating live V. cholerae O1 into the ligated ileal loops. The fate of the vibrios in the intestinal tissues was then studied by transmission electron microscopy at different times after the inoculation. It was concluded that live V. cholerae O1 were initially taken up by the M cells which overlay Peyer's patches and which subsequently delivered the intact vibrios to phagocytic cells in the Peyer's patches. These phagocytic cells processed (digested) the vibrios while the lymphocytes and plasma cells infiltrated around them. During the late period of infection (12-15 hours after inoculation of the vibrios), vibrios were also found passing through the loose intercellular spaces between the absorptive epithelial cells into the underlying intestinal tissues.

Over the past two decades a wide range of immunological studies have been carried out on organisms responsible for causing serious bacterial and parasitic diseases in Thailand. Particular emphasis has been placed on cholera, where investigations have been directed towards vaccine development and rapid diagnosis and on diagnosis of typhoid and enterotoxigenic Escherichia coli. In the area of parasitic diseases emphasis has been directed especially to immunodiagnosis of helminthic diseases, including gnathostomiasis, paragonimiasis, opisthorchiasis, strongyloidiasis and trichinellosis. These studies are reviewed in this report.

Liposomes were prepared from bovine brain sphingomyelin and cholesterol. They were reinforced by incorporation of osmium tetroxide to prevent their immediate degradation inside the host. Combined Vibrio cholerae antigens (lipopolysaccharide, crude cell-bound hemagglutinin and procholeragenoid) were orally administered to experimental rats either as free or liposome-associated. A total of 70 experimental rats was utilized in experiments comparing the immune responses of rats to liposome-associated vaccine, free vaccine, liposomes, or placebo, and to vaccines where the lipid or antigen levels were reduced. Immediately after feeding with sodium bicarbonate to lower the gastric acidity, they were fed either cholera vaccines or placebo. Results from serum ELISA revealed that the liposomes localized the immune response to the intestinal mucosa. They displayed an adjuvant property in terms of evoking a higher immune response to V. cholerae antigens, as measured by the appearance of specific antibody-producing cells in the intestinal mucosa, than when the antigens were fed alone. The adjuvanticity was found to be lipid dose dependent. Liposomes prepared with high lipid content enhanced immunogenicity of the admixture antigens to a greater degree.

Specific antibodies to V. cholerae lipopolysaccharide (LPS), cell-bound haemagglutinin (CHA) and toxin (CT) in the intestinal lavages of healthy Thais and Thai cholera patients during the convalescence period were determined by enzyme-linked immunosorbent assay. Only IgM and IgA specific antibodies were detectable in the specimens. All of the persons who were just recovered from cholera had IgA anti-CT and IgA anti-LPS and 82.4% had IgA anti-CHA. The IgA anti-CT, anti-LPS and anti-CHA were detected also in the gut fluids of 70.6%, 94.1% and 88.2%, respectively, of the healthy controls. The mean levels of the IgA antibodies of all specificities between the two groups of individuals were not different. However, the IgM anti-CT and IgM anti-LPS of the cholera patients increased during the convalescence period. The levels, therefore, were significantly higher than those of the controls. The ratios of IgA anti-CT: IgM anti-CT and IgA anti-LPS: IgM anti-LPS among the patients were 2.93:1 and 2.02:1, respectively while those of the controls were 10:1 and 34:1, respectively. IgA antibodies predominated in the lavages of both groups of the individuals.

Pathological abnormalities of the infant mouse small intestines were evident after V.cholerae El Tor, Ogawa, infection. These were revealed by an enteritis manifested by mononuclear cells containing inflammatory exudate, intervillous blood capillary congestion, marked increase in goblet cells, desquamation of epithelium and diapedesis of cells. Mice received V.cholerae pretreated with high concentration of antihemolysin, though developed diarrhea, had marked decrease in pathological severity. The less amount of the antibodies the more pathological changes were found.

Specific antisera to V. cholerae El Tor hemolysin were prepared. The sera exhibited the following characteristics: formed a single precipitin band in immunoelectrophoresis against the crude preparation of hemolysin, had no passive hemagglutinating antibodies against V. cholerae LPS sensitized cells, possessed neutralizing property to the homologous hemolysin, and afforded some small degree of protection to oral challenge of V. cholerae El Tor in experimental animals.

Albino rats aged 7-8 weeks old purchased from the National Laboratory Animal Centre, Salaya, Nakhon Pathom, were found to be a good animal model for the study on immunogenicity of V. cholerae antigens. Seventy-two rats were fasted for 15 hours before feeding each one with 1 ml of 5% NaHCO3 to reduce gastric acidity prior to immunization. They were divided into 9 groups of 8 rats and immunized orally with 2 ml, each, of the V. cholerae antigens dissolved or suspended in Cassamino acid as follows: group 1 (control): Cassamino acid (Ca) alone; group 2 (control): 2.5% formalinized sheep red blood cells (F-SRBC); group 3: 1,000 micrograms of lipopolysaccharide (LPS); group 4: 100 micrograms of procholeragenoid (P); group 5: 80 haemagglutinating units of cell-bound haemagglutinin (CHA) adsorbed onto the surface of F-SRBC (CH-SRBC); group 6: 500 micrograms of LPS + 50 micrograms of P; group 7: CH-SRBC + 50 micrograms of P; group 8: combined vaccine formula 1 consisted of 500 micrograms of LPS, CH-SRBC and 50 micrograms of P and group 9: combined vaccine formula 2 consisted of 1,000 micrograms of LPS, CH-SRBC and 100 micrograms of P. The immunization was repeated once more 14 days later. Five days, thereafter, the rats were killed and their jejuni were removed for cryostat sectioning. Antibody producing cells against LPS (anti-LPS cells), P (anti-CT cells) and CHA (anti-CHA cells) in the intestinal lamina propria were enumerated by double antibody sandwich method of immunofluorescence using pure LPS, cholera toxin (CT) and pure CHA as the antigens in the assay, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymic fragments of IgG antibody were tested for their protective abilities in the infant mouse cholera model. F(ab')2 retained the full protective activity of the parent IgG molecule despite losses in complement fixation and opsonic functions. Fab' and Fab fragments also contained significant protective activity but at a level of only 10% of the intact IgG or F(ab')2. Self-recombinant univalent F(ab')2 also contained about 10% of the protective activity of the divalent F(ab')2 parent molecule. These results are interpreted as evidence that cross-linking is an important mechanism by which specific antibody protects mucosal surfaces against experimental infection with Vibrio cholerae.

The finding that Fab fragments of the anti-E1 Tor haemagglutinin were able to afford protection in vivo (low but significant), as well as a significant reduction in Vibrio cholerae adherence to isolated intestinal epithelial cells in-vitro, implicate that masking of these cell-bound haemagglutinin sites per se, would be sufficient to confer protection in E1 Tor cholera infection. Subsequently, the related working hypothesis that the E1 Tor cell-bound haemagglutinin is playing an adhesive role is validated. In natural immunity, it could be envisaged that antiserum to this cell-bound haemagglutinin of V. cholerae E1 Tor would be highly protective due to the synergistic effects of the dual protective mechanisms in operation at the intestinal sites viz. a masking and agglutinating phenomenon.

Cross-reactive antibody responses were assessed in volunteers vaccinated with classical Inaba and Ogawa cholera vaccines. The El Tor, Ogawa vibrios, the most often biotype, and serotype found to be the causative agent of cholera in Thailand, or their product were used throughout the in vitro and in vivo tests. The test involved were the passive hemagglutination test, vibriocidal tests and the mouse protection test. Classes of specific immunoglobulins produced in the volunteers were determined using anti-immunoglobulin enhancement of hemagglutination. It was found that the levels of hemagglutinating and vibriocidal antibodies reached their peaks on day 7 after the vaccination and were statistically constant for 3 months. Significant decrease was observed thereafter. The mouse protective antibody titer was highest at 1 month after the vaccination then declined significantly at the 6th month. Classes of specific immunoglobulins were found to be either IgM or IgG alone or mixture of both.

Several antibody preparations were tested for their ability to reduce adsorption of Vibrio cholerae to isolated intestinal epithelial cells, and this ability was related to agglutination and protective activity in infant mice. The results demonstrate that (1) the reduction in adsorption of V. cholerae to epithelial cells correlates with the degree of agglutination for given antibody preparation; (2) intact tantibodies protect infant mice from cholera only at concentrations that agglutinate the bacteria; and (3) purified antibodies to flagellar antigens protect infant mice from cholera. These results indicate that cross-linking of bacteria by antibody causes a reduction in the number of organisms adsorbed to the intestinal wall. Thus antibody cross-linking plays an important role in immunity to cholera in infant mice.

V. cholerae El Tor Ogawa strain O17SR grown on trypticase soy agar were extracted with 0.05 M cyclohexylamino propane sulfonic acid (CAPS) pH 9.5 at 37 degrees C for 1 hour. The bacteria were then removed by centrifugation and millipore filtration. The filtered fluid, after being dialysed against many changes of cold distilled water, was concentrated and passed through Sephadex G200 column. Three protein profiles were eluted out with 0.05 M Tris buffer pH 8.6. The haemagglutinin and the bacterial lipopolysaccharide (LPS) were confined to the first profile. They were subsequently separated by agarose gel electrophoresis. The haemagglutinin was found to be more anodic than the LPS. After homogenization of the gel strips containing the haemagglutinin followed by centrifugation at 9,000 g pure haemagglutinin was obtained in the supernatant. Rabbit aniserum against pure haemagglutinin contained protective antibodies against V. cholerae infection in the baby mouse model. Specific antibodies prepared from this antiserum was as protective as the antibodies directed against whole V. cholerae and heat stable somatic antigens of V. cholerae upon the same weight unit.

Haemagglutinating, vibriocidal and mouse protective antibody responses in cholera patients were found to be maximum on the 7th day of admission. The mouse protective antibody on the first day at the hospital was lower than those of human volunteers. The circulating antibodies in the patients declined to normal levels or lower than normal before 3 months after the acute onset.