Effective Aeromonas specific monoclonal antibody for immunodiagnosis

Objective:To identify the monoclonal antibody specific to Aeromonas spp.,a Gram negative bacteria causing gastroenteritis and wound infection.Methods:The monoclone,namely 88F2-3F4,was produced from hybridoma technology.The specificity of antibody secreted from 88F2-3F4 was tested against other Gram negative bacteria frequently found in gastrointestinal tract.Then the antibody was used for searching Aeromonas antigens in artificial seeded rectal swab cultures by dot-blot enzyme linked immunosorbent assay.Results:88F2-3F4 produced an antibody that recognized an antigen with a molecular mass of 8.5 kDa in all 123 isolates of the seven Aeromonas species tested,but recognized no epitope of any other Gram-negative bacterium typically found in the gastrointestinal tract.A dot-blot enzyme linked immunosorbent assay based on this antibody showed 86.49％ sensitivity and 92.13％ specificity.Conclusions:88F2-3F4 monoclonal antibody could react with all Aeromonas isolates,but not other Gram negative bacteria,therefore it should be a useful tool for the detection of Aeromonas antigen in clinical and environmental samples.

Molecular and immunological analyses of confirmed Plasmodium vivax relapse episodes.

BACKGROUND: Relapse infections resulting from the activation hypnozoites produced by Plasmodium vivax and Plasmodium ovale represent an important obstacle to the successful control of these species. A single licensed drug, primaquine is available to eliminate these liver dormant forms. To date, investigations of vivax relapse infections have been few in number. RESULTS: Genotyping, based on polymorphic regions of two genes (Pvmsp1F3 and Pvcsp) and four microsatellite markers (MS3.27, MS3.502, MS6 and MS8), of 12 paired admission and relapse samples from P. vivax-infected patients were treated with primaquine, revealed that in eight of the parasite populations in the admission and relapse samples were homologous, and heterologous in the remaining four patients. The patients' CYP2D6 genotypes did not suggest that any were poor metabolisers of primaquine. Parasitaemia tended to be higher in the heterologous as compared to the homologous relapse episodes as was the IgG3 response. For the twelve pro- and anti-inflammatory cytokine levels measured for all samples, only those of IL-6 and IL-10 tended to be higher in patients with heterologous as compared to homologous relapses in both admission and relapse episodes. CONCLUSIONS: The data from this limited number of patients with confirmed relapse episodes mirror previous observations of a significant proportion of heterologous parasites in relapses of P. vivax infections in Thailand. Failure of the primaquine treatment that the patients received is unlikely to be due to poor drug metabolism, and could indicate the presence of P. vivax populations in Thailand with poor susceptibility to 8-aminoquinolines.

Leptospira species in floodwater during the 2011 floods in the Bangkok Metropolitan Region, Thailand.

Floodwater samples (N = 110) collected during the 2011 Bangkok floods were tested for Leptospira using culture and polymerase chain reaction (PCR); 65 samples were PCR-positive for putatively non-pathogenic Leptospira species, 1 sample contained a putatively pathogenic Leptospira, and 6 samples contained Leptospira clustering phylogenetically with the intermediate group. The low prevalence of pathogenic and intermediate Leptospira in floodwater was consistent with the low number of human leptospirosis cases reported to the Bureau of Epidemiology in Thailand. This study provides baseline information on environmental Leptospira in Bangkok together with a set of laboratory tests that could be readily deployed in the event of future flooding.

A single injection of 19 kda carboxy-terminal fragment of Plasmodium yoelii merozoite surface protein 1 (PyMSP1(19)) formulated with Montanide ISA and CpG ODN induces protective immune response in mice.

OBJECTIVE: To investigate the efficacy of a vaccine formulation of the 19 kDa conserved carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (PyMSP1(19)) formulated with CpG ODN 1826 and Montanide ISA51 or ISA720 when used to immunize mice by a single injection. METHODS: Groups of BALB/c mice were immunized parenterally with one, two or four injections with PBS or PyMSP1(19) formulated with CpG ODN in ISA51 or ISA720. Sera were collected weekly and assessed for total IgG and IgG subclass titers. Protection was tested by challenge infection with P. yoelii YM. RESULTS: Interestingly, single injection immunization showed the same kinetics of antibody responses as two- or four-injection immunization. However, the peak antibody response induced by PyMSP1(19) in CpG ODN and ISA51 appeared earlier than that induced by PyMSP1(19) in CpG ODN and ISA720 (28 days vs 41 days). At day 63 after the first injection, the PyMSP1(19)-specific IgG antibody levels by single injection and four-injection immunizations were not different. However, the levels of the IgG2a antibody subclass were significantly lower by single injection immunization with PyMSP1(19) in CpG ODN and ISA720. The antibodies were sustained at high levels for at least 20 weeks. After challenge infection, all mice immunized by a single injection of PyMSP1(19) in CpG ODN and ISA51 survived with low-grade parasitemia, while 50% of mice immunized with PyMSP1(19) in CpG ODN and ISA720 died with high levels of parasitemia. CONCLUSION: These findings suggest that MSP1(19) immunization by a single injection can induce protective immunity, particularly when formulated with an appropriate strong adjuvant.

Detection of Salmonella invA gene in shrimp enrichment culture by polymerase chain reaction.

Contamination of seafood with salmonellae is a major public health concern. Detection of Salmonella by standard culture methods is time consuming. In this study, an enrichment culture step prior to polymerase chain reaction (PCR) was applied to detect 284 bp fragment of Salmonella invA in comparison with the conventional culture method in 100 shrimp samples collected from four different shrimp farms and fresh food markets around Bangkok. Samples were pre-enriched in non-selective lactose broth (LB) and selective tetrathionate broth (TTB). PCR detection limit was 10 pg and 10(4) cfu/ml of viable salmonellae with 100% specificity. PCR assay detected 19 different Salmonella serovars belonging to 8 serogroups (B, C1, C2-C3, D1, E1, E4 and K) commonly found in clinical and environmental samples in Thailand. The detection rate of PCR following TTB enrichment (24%) was higher than conventional culture method (19%). PCR following TTB, but not in LB enrichment allowed salmonella detection with 84% sensitivity, 90% specificity and 89% accuracy. Shrimp samples collected from fresh food markets had higher levels of contaminated salmonellae than those from shrimp farms. The results indicated that incorporation of an enrichment step prior to PCR has the potential to be applied for detection of naturally contaminated salmonellae in food, environment and clinical samples.

Haplotypes of IL12B promoter polymorphisms condition susceptibility to severe malaria and functional changes in cytokine levels in Thai adults.

Polymorphic variability in immune response genes, such as IL12B, encoding the IL-12p40 subunit is associated with susceptibility to severe malaria in African populations. Since the role of genetic variation in conditioning severe malaria in Thai adults is largely unexplored, the functional association between IL12B polymorphisms [i.e. IL12Bpro (rs17860508) and IL12B 3' UTR T/G (rs3212227)], severe malaria and cytokine production was examined in patients with Plasmodium falciparum infections (n = 355) recruited from malaria endemic areas along the Thai-Myanmar border in northwest Thailand. Circulating IL-12p40 (p = 0.049) and IFN-gamma (p = 0.051) were elevated in patients with severe malaria, while only IL-12p40 was significantly higher in severe malaria patients with hyperparasitaemia (p = 0.046). Carriage of the IL12Bpro1.1 genotype was associated with enhanced severity of malaria (OR, 2.34; 95% CI, 0.94-5.81; p = 0.066) and hyperparasitaemia (OR, 3.42; 95% CI, 1.17-9.87; p = 0.025) relative to the IL12Bpro2.2 genotype (wild type). Individuals with the IL12Bpro1.1 genotype also had the lowest IL-12p40 (p = 0.002) and the highest IFN-gamma (p = 0.004) levels. Construction of haplotypes revealed that carriage of the IL12Bpro-2/3' UTR-T haplotype was associated with protection against severe malaria (OR, 0.51; 95% CI, 0.29-0.90; p = 0.020) and reduced circulating IFN-gamma (p = 0.06). Thus, genotypic and haplotypic variation at IL12Bpro and IL12B 3' UTR in this population influences susceptibility to severe malaria and functional changes in circulating IL-12p40 and IFN-gamma levels. Results presented here suggest that protection against severe malaria in Thai adults is associated with genotypic variants that condition enhanced IL-12p40 and reduced IFN-gamma levels.

Expression of toll-like receptors on antigen-presenting cells in patients with falciparum malaria.

The continuous release of blood-stage malaria parasites and their products can activate components of the innate immune system and induce the production of proinflammatory cytokines. Toll-like receptors (TLRs) have emerged as pattern-recognition receptors, residing on/in innate immune cells whose function is recognizing specific conserved components on different microbes. The aim of this study was to determine the expression of TLR2, TLR4 and TLR9 on antigen-presenting cells (APCs) in patients with mild and severe forms of falciparum malaria. Healthy individuals were used as controls. Peripheral blood mononuclear cells (PBMCs) were stained with specific monoclonal antibodies (mAbs) to investigate the percentage and the level of TLR expression by flow cytometry. Patients with severe and mild malaria showed increased surface expression of TLR2 and TLR4 on CD14(+)monocytes and myeloid dendritic cells (MDCs) and decreased intracellular expression of TLR9 on plasmacytoid dendritic cells (PDCs), compared to those of healthy controls. A significant decrease in the percentage of circulating CD14(+)monocytes and MDCs expressing TLR2 was found in both severe and mild malaria patients. These findings suggested that TLRs might play role in innate immune recognition in which the differential expression of TLRs on APCs could be regulated by the P. falciparum parasite.

IgG antibody profile to c-terminal region of Plasmodium vivax merozoite surface protein-1 in Thai individuals exposed to malaria.

Naturally acquired immune response to C-terminal region of Plasmodium vivax merozoite surface protein1 (PvMSP1) in 200 individuals with recent clinical episodes of malaria from malaria endemic areas along Thai-Myanmar border in the west and Thai-Cambodia border in the east of Thailand was evaluated by enzyme-linked immunosorbent assay (ELISA). The anti-PvMSP1-IgG antibody was observed in 110 individuals (55%). Among IgG responders, IgG1 coexpressed with IgG3 were the predominant subclasses. The levels of anti-PvMSP1 total IgG, IgG1 and IgG3 antibody response seem to be increased with age although no detectable significant correlation was found (r = 0.004, p = 0.484 for total IgG; r = 0.035, p = 0.386 for IgG1; r = -0.600, p = 0.142 for IgG2; r = 0.077, p = 0.227 for IgG3; r = 0.664, p = 0.051 for IgG4). However, the mean level of specific total IgG was highest in the age group of >40 years. These levels of either specific total IgG or each IgG isotype did not vary among individuals with different malaria episodes. A higher level of specific total IgG, IgG1 and IgG3 antibody response related with the lower of parasitemia density was observed although no significant correlation was found. Our data indicate that individuals exposed to vivax malaria in Thailand developed antibodies to the potential candidate vaccine antigen, PvMSP1 (C-terminal).

Long-lasting protective immune response to the 19-kilodalton carboxy-terminal fragment of Plasmodium yoelii merozoite surface protein 1 in mice.

Merozoite surface protein 1 (MSP1) is the major protein on the surface of the plasmodial merozoite, and its carboxy terminus, the 19-kDa fragment (MSP1(19)), is highly conserved and effective in induction of a protective immune response against malaria parasite infection in mice and monkeys. However, the duration of the immune response has not been elucidated. As such, we immunized BALB/c mice with a standard four-dose injection of recombinant Plasmodium yoelii MSP1(19) formulated with Montanide ISA51 and CpG oligodeoxynucleotide (ODN) and monitored the MSP1(19)-specific antibody levels for up to 12 months. The antibody titers persisted constantly over the period of time without significant waning, in contrast to the antibody levels induced by immunization with Freund's adjuvant, where the antibody levels gradually declined to significantly lower levels 12 months after immunization. Investigation of immunoglobulin G (IgG) subclass longevity revealed that only the IgG1 antibody level (Th2 type-driven response) decreased significantly by 6 months, while the IgG2a antibody level (Th1 type-driven response) did not change over the 12 months after immunization, but the boosting effect was seen in the IgG1 antibody responses but not in the IgG2a antibody responses. After challenge infection, all immunized mice survived with negligibly patent parasitemia. These findings suggest that protective immune responses to MSP1(19) following immunization using oil-based Montanide ISA51 and CpG ODN as an adjuvant are very long-lasting and encourage clinical trials for malaria vaccine development.

Mutation in the rpoB gene of the rifampicin resistant M. avium complex strains from Thailand.

Forms of mutation never before described in the rpoB gene are reported for a sample of 20 rifampicin-resistant Mycobacterium avium Complex (MAC) strains isolated from AIDS patients in Thailand. All strains were analyzed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and polymerase chain reaction-DNA sequencing (PCR-DNA sequencing). Sequence analysis of these strains revealed that only one strain (5%) has missense mutation at Lys-626 (Thr) and the rest of the strains had 15 different silent mutations within a 542 bp region of the rpoB gene. Five strains (25%) had a silent mutation at only one position, 7 (35%) at 2 positions, 7 (35%) at 3 positions, and 1 (5%) at 7 positions. The silent mutation at the Ala-630 codon occurred in the largest proportion of the strains (15 strains, 75%), followed by the Val-581 in 8 strains (40%), Tyr-578 and Thr-600 in 4 strains (20%), and Gly-597 in 3 strains (15%). This investigation demonstrates that mutation in the rpoB gene of MAC strains from Thailand are more varied than previously reported for RIF MAC strains. PCR-SSCP screening clearly separated RIFr strains from rifampicin-susceptible (RIFs) strains.

Detection of P. vivax antigens in malaria endemic populations of Nepal by ELISA using monoclonal antibodies raised against Thai isolates.

An indirect enzyme linked immunosorbent assay (ELISA) using monoclonal antibody (MAb) originated from the native Thai isolates of P. vivax (McPV1) and the polyclonal antibody (PAb) raised against Nepali isolates of P. vivax was developed for detection of P vivax antigens in red cell lysates. The assay was specific (100%) since it was positive only with P. vivax-infected erythrocytes and was negative when erythrocytes from 40 healthy individuals from malaria non-endemic areas and 40 P. falciparum infected erythrocytes were tested. When the assay was applied to 203 vivax blood samples already proven by microscopic examination collected from Dhanusha district of Nepal, and using the cut-off level of the mean optical density (OD) (0.144) of 40 healthy individuals who had been living in malaria-endemic areas (0.073) + 2 SD (0.016), the assay could detect 189/203 samples, indicating the sensitivity of the test was 93.1% with a detection limit of erythrocytes of 240 parasites/10(6) erythrocytes. In addition, the assay was negative when 40 blood samples with fever of unknown origin, collected from the same malaria-endemic areas, were tested. However, there was a significant correlation between OD values and parasitemia (r=0.649; p=0.018). The results indicate that MAb-PAb indirect ELISA using MAb raised against Thai isolates of P. vivax as the coating antibodies, and polyclonal antibodies raised against local Nepali isolates as the detecting antibody, could detect P. vivax antigens with high degrees of sensitivity and specificity. Furthermore, it seems that the McPV1 MAb raised against Thai isolates of P. vivax could recognize the antigens of Nepali isolates in a wide range of blood samples.

Immunity to malaria after administration of ultra-low doses of red cells infected with Plasmodium falciparum.

BACKGROUND: The ability of T cells, acting independently of antibodies, to control malaria parasite growth in people has not been defined. If such was shown to be effective, an additional vaccine strategy could be pursued. Our aim was to ascertain whether or not development of cell-mediated immunity to Plasmodium falciparum blood-stage infection could be induced in human beings by exposure to malaria parasites in very low density. METHODS: We enrolled five volunteers from the staff at our research institute who had never had malaria. We used a cryopreserved inoculum of red cells infected with P falciparum strain 3D7 to give them repeated subclinical infections of malaria that we then cured early with drugs, to induce cell-mediated immune responses. We tested for development of immunity by measurement of parasite concentrations in the blood of volunteers by PCR of the multicopy gene STEVOR and by following up the volunteers clinically, and by measuring antibody and cellular immune responses to the parasite. FINDINGS: After challenge and a extended period without drug cure, volunteers were protected against malaria as indicated by absence of parasites or parasite DNA in the blood, and absence of clinical symptoms. Immunity was characterised by absence of detectable antibodies that bind the parasite or infected red cells, but by the presence of a proliferative T-cell response, involving CD4+ and CD8+ T cells, a cytokine response, consisting of interferon gamma but not interleukin 4 or interleukin 10, induction of high concentrations of nitric oxide synthase activity in peripheral blood mononuclear cells, and a drop in the number of peripheral natural killer T cells. INTERPRETATION: People can be protected against the erythrocytic stage of malaria by a strong cell-mediated immune response, in the absence of detectable parasite-specific antibodies, suggesting an additional strategy for development of a malaria vaccine

Identification of T cell epitopes on the 33-kDa fragment of Plasmodium yoelii merozoite surface protein 1 and their antibody-independent protective role in immunity to blood stage malaria.

Merozoite surface protein 1 (MSP1) of malaria parasites undergoes proteolytic processing at least twice before invasion into a new RBC. The 42-kDa fragment, a product of primary processing, is cleaved by proteolytic enzymes giving rise to MSP1(33), which is shed from the merozoite surface, and MSP1(19), which is the only fragment carried into a new RBC. In this study, we have identified T cell epitopes on MSP1(33) of Plasmodium yoelii and have examined their function in immunity to blood stage malaria. Peptides 20 aa in length, spanning the length of MSP1(33) and overlapping each other by 10 aa, were analyzed for their ability to induce T cell proliferation in immunized BALB/c and C57BL/6 mice. Multiple epitopes were recognized by these two strains of mice. Effector functions of the dominant epitopes were then investigated. Peptides Cm15 and Cm21 were of particular interest as they were able to induce effector T cells capable of delaying growth of lethal P. yoelii YM following adoptive transfer into immunodeficient mice without inducing detectable Ab responses. Homologs of these epitopes could be candidates for inclusion in a subunit vaccine.

Diagnosis of human leptospirosis by monoclonal antibody-based antigen detection in urine.

Hybridomas secreting specific monoclonal antibodies (MAb) to all members of the genus Leptospira (clone LF9) and those that are specific only to the pathogenic species (clones LD5 and LE1) were produced. MAb LF9, which was immunoglobulin G1 (IgG1), reacted to a 38-kDa component of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell lysates of all Leptospira spp., while MAb LD5 and MAb LE1, which were IgG1 and IgG2a, respectively, reacted to the 35- to 36-kDa components of all serogroups of the pathogenic species of LEPTOSPIRA: The MAb LD5 was used in a dot blot-enzyme-linked immunosorbent assay (dot-ELISA) for detecting Leptospira antigen in urine samples serially collected from two groups of patients diagnosed with leptospirosis, i.e., 36 clinically diagnosed patients and 25 Leptospira culture confirmed patients. Their serum samples were tested serologically by IgM Dipstick assay, indirect immunofluorescence assay (IFA), and/or microscopic agglutination test (MAT). Urine samples of 26 patients diagnosed with other illnesses and 120 healthy individuals served as controls. For the first group of patients, who had been ill for an average of 3.4 days before hospitalization, the IgM Dipstick test, IFA, and MAT were positive for 69.4, 70.0, and 85.7% of patients, while the Leptospira antigenuria tested by the MAb-based dot-ELISA was positive for 75.0, 88.9, 97.2, 97.2, and 100% of patients on days 1, 2, 3, 7, and 14 of hospitalization, respectively. All but 1 of 11 patients whose serum samples collected on the first day of hospitalization were IgM seronegative, were positive by urine antigen test on day 1. This is strong evidence that detection of antigen in urine can provide diagnostic information that could be useful in directing early therapeutic intervention. The MAT was positive in 10 of 12 patients (83.3%) of the 25 culture-positive Leptospira patients who had been ill for an average of 5.04 days before hospitalization, and the Leptospira antigen was found in 64.0, 84.0, 96.0, 100, 100, 100, and 100% of the patients' urine samples collected on days 1, 2, 3, 4, 5, 6, and 7 of hospitalization, respectively. Leptospira antigenuria was found in 3 of the 26 patients diagnosed with other illnesses and 1 of the 120 healthy controls. The reasons for this positivity are discussed. The detection of antigen in urine by the monoclonal antibody-based dot-ELISA has high potential for rapid, sensitive, and specific diagnosis of leptospirosis at a low cost.