Flagellar Antigens Research Articles

Production of recombinant antigens, polyclonal antibodies for development of an ELISA assay to detect Salmonella spp. in food

Salmonella is one of the most important food-borne pathogens. Early detection of Salmonella is necessary for food safety and hygiene. We have developed an ELISA assay, using polyclonal antibodies which can detect most Salmonella serotypes. To produce the antibodies, the recombinant flagellar antigens or H antigens of S. Enteritidis (H:g,m) and S. Typhimurium (H:i), were utilized to inject into rabbit, due to the fact that they are immunodominant antigenic surface structures of Salmonella and represent for two groups of the most common H antigen types, α cluster and G complex (McQuiston, J. R. et al., 2004). The genes coding for antigens (fliCgm and fliCi) were cloned into vector pGEX-5X-1 and expressed in the strain E. coli BL21(DE3). The recombinant E. coli was cultured in 200ml LB and the expression of fliCgm and fliCi was induced with 0.4mM IPTG. 2.5mg of each GST-fused protein (GST-H:g,m and GST-H:i) was harvested after purifying by GSTrap FF column. The protein was confirmed by SDS-PAGE analysis and Western Blot with the specific anti-H antigen antibody. 100μg of protein in complete Freund adjuvant was injected to rabbit. Blood collection and booster injection were done every 3 weeks. Terminal blood collection was at the fourth booster injection. 100ml of antiserum of each H antigen was obtained and was run through protein A column to purify polyclonal antibody. Polyclonal antibody was successfully conjugated to HRP. The sandwich ELISA procedure utilized the mixture of antibodies. After optimization, the concentration of the mixture of antibodies in the sandwich ELISA method was 250ng of capture antibodies and 100ng of antibody-HRP conjugate. The optimized sandwich ELISA protocol was applied to test 60 Salmonella strains isolated from food samples and check the cross-reactivity with some other bacteria in food. The result showed that 99% of samples was positive of Salmonella presence (OD492>0.3), and only 1% was negative (OD492<0.3). Sandwich ELISA of some other bacteria in food, including E. coli, Shigella spp., Vibrio parahaemolyticus, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus, resulted in very low values of OD492 (<0.2). In conclusion, the ELISA assay was confirmed the specificity to Salmonella sp. and a very low cross-reactivity to other bacteria presenting in food.

Population-based study of diagnostic assays for Borrelia infection: comparison of purified flagella antigen assay (Ideia™, Dako Cytomation) and recombinant antigen assay (Liaison®, DiaSorin)

BackgroundTesting for Borrelia-specific IgM and IgG-antibodies are often performed on a variety of poorly defined symptoms, and isolated IgM results are a frequent finding, which results in diagnostic uncertainty and further testing. We wanted to test the hypothesis that Borrelia-specific assays using recombinant antigens perform differently from assays based on purified flagella antigen.MethodsWe compared the use of recombinant antigens (LIAISON® DiaSorin, Saluggia, Italy) and purified flagella antigen (IDEIA™ Borrelia, DakoCytomation, Glostrup, Denmark) in the assay for Borrelia-specific IgM and IgG-antibodies. The assays were tested on an unselected population of serum samples submitted from general practice. A total of 357 consecutive samples for analysis of Borrelia IgM and IgG antibodies. Furthermore, we analysed 540 samples for Borrelia-specific IgM or IgG antibodies first by the IDEIA™ and, if they were positive, the samples were further analysed using the LIAISON® assay. To verify the correctness of the patient's serological status, discrepant samples were analysed by line blots (EcoLine, Virotech).ResultsIn the consecutive series of 357 samples, the IgM assays detected 308 negative and 3 positive samples with concordant results. Compared with the line blot, the IDEIA™ system produced 21 false-positive IgM results, whereas the LIAISON® system produced only one false-positive IgM result. The IgG assays showed 1 positive and 328 negative concordant results. The LIAISON® system produced 9 true IgG-positive samples that were not detected by the IDEIA™ system, but the former produced 4 positive IgG results that were negative by line blot.ConclusionDiagnostic assays based on flagella antigen seem to show more false-positive IgM and false-negative IgG results than assays based on recombinant antigens. The latter may reduce the number of presumably false-positive IgM results and identify more IgG-positive subjects, but this system also produces more false-positive IgG results.

Bacteria and bacterial rRNA genes associated with the development of colitis in IL-10−/− Mice

Microorganisms appear to play important yet ill-defined roles in the etiology of inflammatory bowel disease (IBD). This study utilized a novel population-based approach to identify bacteria and bacterial rRNA genes associated with the development of colitis in IL-10(-/-) mice. Mice were housed in 2 environments: a community mouse facility where the mice were fed nonsterile chow (Room 3) and a limited access facility where the mice were fed sterile chow (Room 4). Every month the disease activity levels were assessed and fecal bacterial compositions were analyzed. At the end of the experiments histological and bacterial analyses were performed on intestinal tissue. Although disease activity increased over time in both environments, it progressed at a faster rate in Room 3 than Room 4. Culture and culture-independent bacterial analyses identified several isolates and phylotypes associated with colitis. Two phylotypes (GpC2 and Gp66) were distinguished by their negative associations with disease activity in fecal and tissue samples. Notably, rRNA genes from these phylotypes had high sequence identity (99%) to an rRNA gene from a previously described flagellated Clostridium (Lachnospiraceae bacterium A4). The negative associations of these 2 phylotypes (GpC2 and Gp66) suggest that these bacteria were being immunologically targeted, consistent with prior findings that the Lachnospiraceae bacterium A4 bears a prevalent flagellar antigen for disease-associated immunity in murine immune colitis and human Crohn's disease. Identification of these associations suggests that the experimental approach used in this study will have considerable utility in elucidating the host-microbe interactions underlying IBD.

PCR Method To Identify Salmonella enterica Serovars Typhi, Paratyphi A, and Paratyphi B among Salmonella Isolates from the Blood of Patients with Clinical Enteric Fever

PCR methodology was developed to identify Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B. One multiplex PCR identifies serogroup D, A, and B and Vi-positive strains; another confirms flagellar antigen "d," "a," or "b." Blinded testing of 664 Malian and Chilean Salmonella blood isolates demonstrated 100% sensitivity and specificity.

The continuing evolution of a bacterial pathogen

Although Escherichia coli is a prototypic commensal bacterial species of the mammalian intestine and a laboratory workhorse for molecular biology, certain strains of this species are capable of causing significant human disease. The spectrum of disease caused by E. coli includes enteric/diarrheal disease, urinary tract infections, renal failure, and sepsis/meningitis (1). The different pathotypes of E. coli possess genes encoding a wide variety of virulence factors that are frequently encoded on mobile genetic elements such as bacteriophages, plasmids, and pathogenicity islands. E. coli strains can be serotyped as one of >10,000 possible combinations of O (LPS) and H (flagellar) antigens, but one serotype, O157:H7, has achieved particular notoriety as a cause of deadly outbreaks of food-borne illness throughout the world. The work of Manning et al. (2) in this issue of PNAS reveals significant new information on the evolution and genetic composition of E. coli strains belonging to the O157:H7 serotype and reports the emergence of a new variant that appears to cause more severe disease.

Isolation of Escherichia coli O157:H7 from ground beef samples collected from beef markets, using conventional culture and polymerase chain reaction in Mashhad, northeastern Iran

Summary Escherichia coli O157:H7 is an important human pathogen causing haemorrhagic colitis, haemolytic- uraemic syndrom and thrombotic thrombocytopenic purpura. In this study, 100 ground beef samples were collected randomly from beef markets in June 2004. For isolation of the bacteria, samples were firstly enriched in modified trypticase soy broth, followed by plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Consequently, the suspected non-sorbitol fermenting (NSF) colonies were confirmed by biochemical tests and employed for polymerase chain reaction (PCR) assay, using primers specific for O157 and H7 antigens gene. In this study, 7 NSF E. coli colonies were isolated; in PCR assay only one of them confirmed as E. coli O157:H7. The PCR assay employed in this study may be a possible alternative to immunological assays which detects somatic and flagellar antigens.

Nationwide Surveillance of salmonella in the Faeces of Pigs in Japan

The prevalence of faecal carriage of salmonella in 5393 pigs reared on 218 pig farms located in 31 of 47 prefectures in Japan over the period July 2003 to June 2005 was investigated. We isolated 172 strains belonging to 20 serovars and one untypable Salmonella enterica from 169 pig faecal samples (3.1%) collected from 48 farms (22.0%). The most prevalent type of S. enterica was untypable O4,12:d:- which lacks phase 2 flagellar antigen, representing 29.1% (50/172) of all isolates. Of 26 S. enterica serovar Typhimurium isolates, 16 strains appeared to be definitive phage type 104 (DT104) by polymerase chain reaction.

Improved Laboratory Diagnostics of Lyme Neuroborreliosis in Children by Detection of Antibodies to New Antigens in Cerebrospinal Fluid

Laboratory diagnostics in Lyme neuroborreliosis need improvement. We hereby investigate 4 new recombinant or peptide Borrelia antigens in cerebrospinal fluid in children with neuroborreliosis to evaluate their performance as diagnostic antigens. An enzyme-linked immunosorbent assay was used to detect IgG antibodies to recombinant decorin binding protein A (DbpA), BBK32, outer surface protein C (OspC), and the invariable region 6 peptide (IR6). The recombinant antigens originated from 3 pathogenic subspecies; Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto. Cerebrospinal fluid and serum from children with clinical features indicative for neuroborreliosis (n = 57) were analyzed. Classification of patients was based on clinical symptoms and laboratory findings. Controls were children with other neurologic diseases (n = 20) and adult patients with no proven infection (n = 16). Sensitivity for DbpA was 82%, for BBK32 70%, for OspC 58% and for IR6 70%. Specificities were 94%, 100%, 97%, and 97%, respectively. No single antigen was superior. When new antigens were combined in a panel, sensitivity was 80% and specificity 100%. The reference flagella antigen showed a sensitivity of 60% and a specificity of 100%. Over all, the B. garinii related antigens dominated. Recombinant DbpA and BBK32 as well as the peptide antigen IR6 perform well in laboratory diagnostics of neuroborreliosis in children. New antigens seem to improve diagnostic performance when compared with the routine flagella antigen. If different antigens are combined in a panel to cover the antigenic diversity, sensitivity improves further and a specificity of 100% can be achieve.

Cellular immune response from Chagasic patients to CRA or FRA recombinant antigens of Trypanosoma cruzi.

We propose to analyze the relation between the cellular immune response of Chagas' disease patients after in vitro stimulation of peripheral blood mononuclear cells (PBMC) with recombinant antigens cytoplasmatic repetitive antigen (CRA) or flagellar repetitive antigen (FRA) of T. cruzi and the chronic clinical forms of disease. Cells were stimulated using phytohemagglutinin, CRA, FRA, or a soluble antigen of Epimastigota (Ag-Epi) for 24 hr, 72 hr, or 6 days. The proliferation of cells was evaluated after 6 days of culture by quantification of incorporated 3H-thymidine. Cytokines were measured in the supernatants obtained after 24 hr (tumor necrosis factor [TNF]-alpha and interleukin [IL]-4), 72 hr (IL-10), and 6 days (interferon [IFN]-gamma) using enzyme-linked immunosorbent assay (ELISA). Cells of the Chagas patients stimulated with the recombinant antigens exhibited higher proliferation responses compared with that of non-Chagas (NC) individuals. However, when proliferation was compared between patients with the cardiac form (CF) or indeterminate form (IF), it was not possible to establish a difference in the response. So far as the cytokines secreted in the culture supernatants after stimulation in vitro with T. cruzi antigens were concerned, the results showed that CRA, as well as Epi-Ag, were able to stimulate the production of TNF-alpha and IFN-gamma in Chagas patients as compared with NC individuals. However, the cytokine levels after stimulation with the T. cruzi antigens were not different between the patients with CF and IF. CRA was capable of inducing a T helper type 1 (Th1) immune response, with elevated production of TNF-alpha and IFN-gamma in Chagas patients that are carriers of CF and IF clinical forms.

Diversity of Bacillus thuringiensis strains from soil in China and their pesticidal activities

A large number of Bacillus thuringiensis (Bt) isolates have been obtained from soil samples in China. The flagellar antigen serotypes, cry genes and crystal proteins of 570 Bt isolates were determined, and the pesticidal activity was assayed against the insects, Plutella xylostella, Heliothis armigera, Phaedon brassicae and Locusta migratoria manilensis, and the snail, Oncomelania hupensis. The results indicated that the Bt isolates were distributed within 35 H-serotypes, in which isolates of H3 were the most abundant (20%) followed by H5 (13%), H7 (9%) and H4 (8.7%), whereas isolates of other H-serotypes were less than 6%. The percentage of isolates containing the genes cry1Ac, cry1Aa/ cry1Ac, cry1Aa/cry1Ac/cry1Ab, and cry1Aa/cry1Ac/cry1C was 14.7%, 6.6%, 5.6%, and 7.0%, respectively, while 265 isolates, representing 46.5% of the 570 Bt isolates, did not show any amplification product for the genes cry1Aa, cry1Ac, cry1C, cry2, cry3, cry4, cry7Aa. Some of the 570 Bt isolates caused high mortality of the assayed pests with 14.9%, 6%, 1.6%, 1.1%, and 0.2% of the isolates killing more than 90% of P. xylostella, H. armigera, P. brassicae, O. hupensis, and L. migratoria manilensis, respectively. The remaining 76.2% of the 570 isolates caused no mortality or less than 90% mortality against the tested insect and snail species.

A linear plasmid truncation induces unidirectional flagellar phase change in H:z66 positive Salmonella Typhi

The process by which bacteria regulate flagellar expression is known as phase variation and in Salmonella enterica this process permits the expression of one of two flagellin genes, fliC or fljB, at any one time. Salmonella Typhi (S. Typhi) is normally not capable of phase variation of flagellar antigen expression as isolates only harbour the fliC gene (H:d) and lacks an equivalent fljB locus. However, some S. Typhi isolates, exclusively from Indonesia, harbour an fljB equivalent encoded on linear plasmid, pBSSB1 that drives the expression of a novel flagellin named H:z66. H:z66+S. Typhi isolates were stimulated to change flagellar phase and genetically analysed for the mechanism of variation. The phase change was demonstrated to be unidirectional, reverting to expression from the resident chromosomal fliC gene. DNA sequencing demonstrated that pBSSB1 linear DNA was still detectable but that these derivatives had undergone deletion and were lacking fljAz66 (encoding a flagellar repressor) and fljBz66. The deletion end-point was found to involve one of the plasmid termini and a palindromic repeat sequence within fljBz66, distinct to that found at the terminus of pBSSB1. These data demonstrate that, like some Streptomyces linear elements, at least one of the terminal inverted repeats of pBSSB1 is non-essential, but that a palindromic repeat sequence may be necessary for replication.

Development of a 5'-nuclease PCR assay for the identification of Escherichia coli strains expressing the flagellar antigen H21 and their detection in food after enrichment

To develop a real-time PCR assay targeting the Escherichia coli flagellar antigen H21 for identification and surveillance of clinically important Shiga toxin-producing E. coli (STEC) serotypes classified in seropathotype C. The fliC allele of STEC O91:H21 strain B2F1 was amplified and sequenced. The nucleotide sequence obtained was compared with fliC genes of E. coli O157:H21, O8:H21 and O113:H21 strains. A pair of oligonucleotide primers and a TaqMan minor groove binder probe specific for fliC-H21 were designed and used in a 5'-nuclease PCR assay. This method was evaluated using a panel of 138 diverse bacterial strains and was shown to be 100% specific for H21. PCR amplification of fliC-H21 from one cell per reaction mixture was possible, and an initial inoculum of 10 STEC H21 colony-forming units per 25 g of ground beef was detected after overnight enrichment. The PCR assay developed was found to be highly sensitive and specific for the identification and detection of E. coli H21 strains in ground beef. The real-time PCR assay targeting the H21 flagellar antigen described here offers a valuable method for the rapid detection and molecular typing of pathogenic STEC H21 strains in food.

Antigen biochips verify and extend the scope of antibody detection in Lyme borreliosis

The antibody response of serum IgM and IgG of patients with neuroborreliosis and erythema migrans of Lyme borreliosis (LB) was examined against a 41-kDa flagellar antigen and an 8-mer synthetic OspC8 peptide (VAESPKKP) derived from the C-terminus of outer surface protein C (OspC) from Borrelia garinii. We developed a streptavidin-modified biochip-based immunodiagnosis and compared it with conventional methods such as enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). The diagnostic sensitivity of the coated biochips was demonstrated to be identical, and the results of conventional assays such as ELISA and WB were confirmed. Flagellar antigens lead to better diagnosis because of a higher discriminative value. By contrast, OspC8, a peptide derived from the outer surface antigen, is less sensitive to identify immunity in LB. The inferior antigenicity of OspC8 may be due to epitope masking. Overall, this system is open to simultaneously analyze a larger family of peptides differing in length. Thus, an array approach is generally more advantageous to extend the pattern of antigens to be tested for antigenicity in LB. Serial analysis during ongoing disease may be valuable to learn more about the course of the disease and intermittent reactivation of infection. Protein biochip as a potential substitution of ELISA and WB method offers the opportunity to study serum immunity in a multiplicity of patients simultaneously.

Novel PCR assay for identification of Salmonella enterica serovar Infantis

We developed, optimized and tested two novel PCR assays specific for Salmonella enterica subspecies enterica serovar Infantis. The fljB gene was chosen as the target sequence. Primers were designed on a consensus sequence built by sequencing the fljB gene of five genetically unrelated Hungarian S. Infantis strains and using sequence data from the GenBank (http://www.ncbi.nih.gov). Two alternative assays were designed, which share the reverse primer. Both proved to be highly specific to S. Infantis, neither reacted with 42 other nontyphoidal serovariants tested. The detection limit of the assays was determined to be 10(5) CFU ml(-1) from pure culture, and 10(6) CFU g(-1) from artificially spiked chicken faeces samples. Although the detection limit is rather high to allow for using them for direct detection, the assays may be useful in identification of S. Infantis both for diagnostic and for research purposes. The described PCR assays allow for the correct identification of S. Infantis even when traditional serotyping methods fail because lack of expression of flagellar antigens.

Serodiagnosis of Salmonella enterica serovar Typhi and S. enterica serovars Paratyphi A, B and C human infections

The aim of this study was to evaluate an immunoassay for the detection of human serum antibodies to the LPS and flagellar antigens of Salmonella Typhi and Salmonella Paratyphi A, B and C, and to the Vi capsular polysaccharide of S. Typhi and S. Paratyphi C. A total of 330 sera were used; these originated from 15 patients who were culture-positive for S. Typhi and 15 healthy controls, together with 300 sera submitted to the Laboratory of Enteric Pathogens for Salmonella serodiagnosis. By SDS-PAGE/immunoblotting, all 15 sera from culture-positive patients had serum antibodies to the 9,12 LPS antigens and 10 had antibodies to the 'd' flagellar antigens. Of the 300 reference sera, 22 had antibodies to the 9,12 LPS antigens, one to the 1,4,5,12 LPS antigens and 12 to the 6,7 LPS antigens. Only two sera had antibodies to flagellar antigens, one of which bound to the 'b' and the other to the 'd' antigen. An ELISA was developed that successfully detected serum antibodies to the Vi capsular polysaccharides, but because of the kinetics of serum antibody production to the Vi, these antibodies may be of limited value in the serodiagnosis of acute infection with S. Typhi and S. Paratyphi C. The immunoassays described here provide a sensitive means of detecting serum antibodies to the LPS, flagellar and Vi antigens of S. Typhi and S. Paratyphi, and constitute a viable replacement for the Widal assay for the screening of sera. The Salmonella serodiagnosis protocols described here are the new standard operating procedures used by the Health Protection Agency's National Salmonella Reference Centre based in the Laboratory of Enteric Pathogens, Colindale, UK.

Single-Nucleotide-Polymorphism Typing and Genetic Relationships of Salmonella enterica Serovar Typhi Isolates

Salmonella enterica serovar Typhi is a clone with a low level of variation. We developed a molecular typing method for serovar Typhi using 38 genome-wide single-nucleotide polymorphisms (SNPs) as markers detected by PCR-restriction enzyme digestion. The 73 worldwide serovar Typhi isolates studied were separated into 23 SNP profiles and four distinct genetic groups. Serovar Typhi isolates expressing the unique flagellar antigen z66 were found to cluster together and branch off from the ancestral group, suggesting that serovar Typhi was initially monophasic with only an H1 antigen and subsequently gained the z66 antigen. Typing using the 38 SNPs gave a discriminatory power of 0.87, and a minimum of 16 SNPs may be used to achieve the same level of differentiation. The SNP typing method we developed will be a valuable tool for global epidemiology studies of serovar Typhi.

Methodologies towards the development of an oligonucleotide microarray for determination of Salmonella serotypes

A DNA-based microarray designed to detect somatic (O) and flagellar (H) antigens present in the five most commonly isolated Salmonella serovars within Canada was developed as an alternative to the traditional Kauffmann–White serotyping scheme currently used to serotype salmonellae. Short oligonucleotide probes were designed based on publicly available sequence data of selected genes responsible for O and H antigen biosynthesis. These targets included: antigen-specific sequences within the flagella (H) antigen phase 1 ( fliC) and phase 2 ( fljB) genes and somatic (O) antigen biosynthesis genes within the rfb cluster (Groups B— rfbJ, C1— wbaA, C2— rfbJ, D1— rfbS). A prototype microarray with 117 O and H antigen-specific probes and controls was used to assess probe performance against two pools of gene target PCR amplicons. A set of 31 of these antigen-specific probes (8 O and 23 H) with high specific signal and low non-specific signal were selected based on t-test ( p-value < 0.01) and log 2 ratio distribution analysis to create a prototype microarray. The microarray was tested against 16 Salmonella strains of known serotype. Based on the strains tested in this study, these probes successfully identified and differentiated 11 of the 12 antigens targeted. The prototype DNA-based typing microarray described here has the potential to be an automated alternative to the traditional antigen–antibody serotyping scheme currently used for Salmonella.

Novel indirect enzyme-linked immunosorbent assay (ELISA) method to detect Total E. coli in water environment

In order to establish ELISA (enzyme-linked immunosorbent assay) method to detect Total E. coli in water environment, E. coli multi-characters antigens in water environment were prepared according to the characters of kinds of E. coli serotypes, including antigen of whole cell, antigen of disrupted whole cell, somatic antigen, flagellar antigen and fimbrial antigen. Total E. coli polyclonal antibodies were obtained from the New Zealand rabbits immunized with these five antigens, respectively. Antibodies generated in this research are with high titers and good purity, can conjugate with antigens, specifically, stably and strongly. Indirect ELISA shows the titers of antibody of whole cell and antibody of disrupted whole cell are both over 1 × 10 5. The cross-reactivity of the antibody is from 12 to 30% which indicate the specificity of the antibody against Total E. coli. Based on these antibodies, we established indirect ELISA method to detect Total E. coli in water environment. The matrix effects were studied and the results show that there is no significant influence by all the factors. The ELISA result shows that the detection limitation could be 10 4 CFU (colony forming units) L −1. The indirect ELISA method developed in this study is well suited for Total E. coli analysis in real water samples as a rapid screen method.

Prevalence of Salmonella antibodies among goats slaughtered for chevon in Bareilly (Northern India)

We screened serum samples of 1024 goats slaughtered for chevon in Bareilly in Northern India for Salmonella antibodies with indirect ELISA, MAT-H (microagglutination test using flagellar antigens e, n, x and 1, 5) and MAT-O (microagglutination test using somatic antigens 4, 12 and 3, 10, 15). Salmonella antibodies were detected in 48, 8 and 40%, goats using Salmonella-cytotoxi-I ELISA, MAT ‘H’ and MAT ‘O’, respectively. After adjusting for test accuracy, the seroprevalence were highest for Salmonella-cytotoxi-I ELISA (46%) followed by agglutinins against ‘O’ 3, 10, 15 (15%) and negligible for other agglutinins. With all 5 tests, prevalence of Salmonella antibodies was significantly higher in females than in males. No significant difference was evident in prevalence of Salmonella antibodies to different antigens in different age groups of male goats except for e, n, x agglutinins that were significantly more prevalent in young adult (<6–18 months) males than in adult (>18 months of age) or young (≤6 months of age) goats. On the other hand, in females, prevalence of Salmonella-cytotoxin-I antibodies and e, n, x agglutinins differed significantly among three age groups, being the most prevalent in adult goats. As expected, the results of different tests had little or no correlation because the different tests targeted antibodies to different antigens.

6th International Conference on Typhoid Fever and Other Salmonelloses, Guilin, China, 12-14 November 2005

The International Conference on Typhoid Fever and Other Salmonelloses is organized every two to three years and has usually taken place in Asia. The first meeting was held in Malaysia 15 years ago. The 6th international meeting, held in Guilin, China, in November 2005, was organized by the Guangxi Centre for Disease Prevention and Control, in collaboration with the World Health Organization (WHO), the International Vaccine Institute (IVI), and the American Society for Microbiology (ASM). Two hundred fifty participants from 27 countries, including Africa, participated. Nine sessions covering the major topics of interest provided the latest updated information on typhoid fever. These include: Epidemiology and disease burden; Molecular pathogenesis and genomics; Contemporary issues in vaccine development; Diagnostics in typhoid fever; Antimicrobial resistance in typhoid fever; Clinical and therapeutic aspects; Socio-behavioural research; Other salmonelloses-related contemporary issues; and a Roundtable on Expanding Vaccine Implementation in Developing Countries. Travel grants for participants from developing countries were supported by the WHO, IVI, and the ASM, and by the Conference Organizing Committee. Epidemiology and disease burden During the epidemiology session, general consensus was raised that typhoid fever remains an important, but under-estimated, disease which has high mortality in developing countries, particularly in Asia. However, there are extremely limited data from Africa, and the burden of disease is probably under-estimated in this region. Endemic disease in South-East Asia is observed to be high, although it varies greatly in different settings. In addition, disease in the paediatric population was higher than previously recognized. The latest published figure for the estimated number of cases is approximately 22 million per year, with an estimated 210,000 deaths (1). This number of deaths was calculated from a conservative figure using a case-fatality rate (CFR) of 1%. However, the reported CFR from presentations during the meeting was generally much higher than this in different studies, with the general agreement that this 1% estimate was under-estimated and that additional robust studies are required. It was suggested that WHO should convene a consultative meeting to resolve this during 2006. Second, the increasing number of Salmonella Paratyphi A isolates recovered in Asia, particularly in China and Pakistan, was noted with concern. In Guingxi province of China, 94% of Salmonella isolates are currently S. Paratyphi A. This situation raised the point of accelerating the development of S. Paratyphi A vaccine candidates. Molecular pathogenesis and genomics Developments in molecular pathogenesis and genomics highlighted the difficulties that surround the serotyping of Salmonella species and the classification of Salmonella strains. The full genome sequence of Salmonella strains indicates that the S. Typhi and S. Paratyphi A have a recent evolutionary history. Using this sequence data, a novel system based on multi-locus sequences typing (MLST) has been devised which should help type Salmonella strains in a more precise approach and could replace classical serotyping in future. New data from Viet Nam showed that a haplotype of the TNF region was associated with fever-clearance time in patients. Individuals who have the protective haplotype (*12*22**1) cleared their fever significantly faster than individuals who did not have this haplotype. Better understanding of the organization of Salmonella pathogenicity islands and diversity of Typhi flagellar antigen expression were presented. Finally, it was recommended that further research was required on (a) S. Typhimurium in Africa, (b) paediatric versus adult strains of S. Typhi, and (c) molecular epidemiology of outbreaks versus endemic strains. Contemporary issues in vaccine development Besides the currently-licensed and available vaccines (Vi polyssacharide and Ty21a), other candidate vaccines, consisting of either subunit vaccine or live-attenuated vaccines, were described. …