Identification and Characterization of a Large Effect QTL from Oryza glumaepatula Revealed Pi68(t) as Putative Candidate Gene for Rice Blast Resistance.

BACKGROUND: Field resistance is often effective and durable as compared to vertical resistance. The introgression line (INGR15002) derived from O. glumaepatula has proven broad spectrum field resistance for both leaf and neck blast. RESULTS: Quantitative Trait Loci (QTL) analysis of INGR15002, led to the identification of two major QTL - qBL3 contributing about 34% and 32% phenotypic variance towards leaf and neck blast resistance, respectively and qBL7 contributing about 25% of phenotypic variance for leaf blast. Further, qBL3 was fine mapped, narrowed down to 300 kb region and a linked SNP maker was identified. By combining mapping with microarray analysis, a candidate gene, Os03g0281466 (malectin-serine threonine kinase), was identified in the fine mapped region and named as Pi68(t). The nucleotide variations in the coding as well as upstream region of the gene was identified through cloning and sequence analysis of Pi68(t) alleles. These significant variations led to the non-synonymous changes in the protein as well as variations (presence/absence) in four important motifs (W-box element; MYC element; TCP element; BIHD1OS) at promoter region those are associated with resistance and susceptible reactions. The effect of qBL3 was validated by its introgression into BPT5204 (susceptible variety) through marker-assisted selection and progeny exhibiting resistance to both leaf and neck blast was identified. Further, the utility of linked markers of Pi68(t) in the blast breeding programs was demonstrated in elite germplasm lines. CONCLUSIONS: This is the first report on the identification and characterization of major effect QTL from O. glumaepatula, which led to the identification of a putative candidate gene, Pi68(t), which confers field resistance to leaf as well as neck blast in rice.

Evaluation of anti-inflammatory effect of Varanadi Kashayam (decoction) in THP-1-derived macrophages.

BACKGROUND: Varanadi Kashayam is an Ayurvedic polyherbal decoction containing 16 ingredients, for which the mechanisms of action involved in controlling chronic inflammatory conditions have not been evaluated. The inhibition of release of proinflammatory cytokines by lipopolysaccharide (LPS)-stimulated monocytes/macrophages is an ideal in vitro model for identifying anti-inflammatory molecules. AIM: The aim of the study is to determine the anti-inflammatory effect of Varanadi Kashayam in THP-1-derived macrophages. MATERIALS AND METHODS: The efficacy of Varanadi Kashayam on monocyte cell differentiation was determined by quantitative polymerase chain reaction to assess the expression of differentiation markers MMP-9, CD36, CD11b and CD14. Further Varanadi Kashayam treated THP-1 macrophages were induced with LPS and the production of proinflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1ß) were measured and corresponding genes expressions were quantified. RESULTS: The results indicate that Varanadi Kashayam reduced the differentiation of THP-1 monocytes to macrophages and downregulated the expression of cell surface markers. Furthermore, it could decrease the release of proinflammatory cytokines from LPS-induced THP-1 macrophages and downregulated the expression of TNF-α and IL-1ß genes. CONCLUSION: The results obtained from this study suggest a possible mechanism of action of the herbal decoction in inflammatory processes and opens up the possibilities of identifying bioactive lead molecules with anti-inflammatory potentials.

Diverse Rice Landraces of North-East India Enables the Identification of Novel Genetic Resources for Magnaporthe Resistance.

North-East (NE) India, the probable origin of rice has diverse genetic resources. Many rice landraces of NE India were not yet characterized for blast resistance. A set of 232 landraces of NE India, were screened for field resistance at two different hotspots of rice blast, viz., IIRR-UBN, Hyderabad and ICAR-NEH, Manipur in two consecutive seasons. The phenotypic evaluation as well as gene profiling for 12 major blast resistance genes (Pitp, Pi33, Pi54, Pib, Pi20, Pi38, Pita2, Pi1, Piz, Pi9, Pizt, and Pi40) with linked as well as gene-specific markers, identified 84 resistant landraces possessing different gene(s) either in singly or in combinations and also identified seven resistant landraces which do not have the tested genes, indicating the valuable genetic resources for blast resistance. To understand the molecular diversity existing in the population, distance and model based analysis were performed using 120 SSR markers. Results of both analyses are highly correlated by forming two distinct subgroups and the existence of high diversity (24.9% among the subgroups; 75.1% among individuals of each subgroup) was observed. To practically utilize the diversity in the breeding program, a robust core set having an efficiency index of 0.82 which consists of 33 landraces were identified through data of molecular, blast phenotyping, and important agro-morphological traits. The association of eight novel SSR markers for important agronomic traits which includes leaf and neck blast resistance was determined using genome-wide association analysis. The current study focuses on identifying novel resources having field resistance to blast as well as markers which can be explored in rice improvement programs. It also entails the development of a core set which can aid in representing the entire diversity for efficiently harnessing its properties to broaden the gene pool of rice.

Field level evaluation of rice introgression lines for heat tolerance and validation of markers linked to spikelet fertility.

Rice lines derived from wild species and mutants can serve as a good resource for favorable alleles for heat tolerance. In all, 48 stable lines including 17 KMR3/O. rufipogon introgression lines (KMR3 ILs), 15 Swarna/O. nivara ILs (Swarna ILs) along with their parents, Nagina 22 (N22) and its 4 EMS induced mutants and 7 varieties were evaluated for heat tolerance under irrigated conditions under field in two seasons, wet season 2012 using poly cover house method and dry season 2013 using late sown method. Spikelet fertility (SF), yield per plant (YP) and heat susceptibility index (HSI) for these two traits were considered as criteria to assess heat tolerance compared to control. Four KMR3 ILs and eight Swarna ILs were identified as heat tolerant based on SF and YP and their HSIs in both wet and dry seasons. S-65 and S-70 showed low SF and high YP consistently in response to heat in both seasons. We provide evidence that SF alone may not be the best criterion to assess heat tolerance and including YP is important as lines with low SF but high YP and vice versa were identified under heat stress. Out of 49 SSR markers linked to spikelet fertility, 18 were validated for five traits. RM229 in wet season and RM430 and RM210 in dry season were significantly associated with both SF and its HSI under heat stress. RM430 was also significantly associated with both YP and its HSI in dry season. Thirty two candidate genes were identified close to nine markers associated with traits under heat stress.

Nucleotide variation and identification of novel blast resistance alleles of Pib by allele mining strategy.

Pib is one of significant rice blast resistant genes, which provides resistance to wide range of isolates of rice blast pathogen, Magnaporthe oryzae. Identification and isolation of novel and beneficial alleles help in crop enhancement. Allele mining is one of the best strategies for dissecting the allelic variations at candidate gene and identification of novel alleles. Hence, in the present study, Pib was analyzed by allele mining strategy, and coding and non-coding (upstream and intron) regions were examined to identify novel Pib alleles. Allelic sequences comparison revealed that nucleotide polymorphisms at coding regions affected the amino acid sequences, while the polymorphism at upstream (non-coding) region affected the motifs arrangements. Pib alleles from resistant landraces, Sercher and Krengosa showed better resistance than Pib donor variety, might be due to acquired mutations, especially at LRR region. The evolutionary distance, Ka/Ks and phylogenetic analyzes also supported these results. Transcription factor binding motif analysis revealed that Pib (Sr) had a unique motif (DPBFCOREDCDC3), while five different motifs differentiated the resistance and susceptible Pib alleles. As the Pib is an inducible gene, the identified differential motifs helps to understand the Pib expression mechanism. The identified novel Pib resistant alleles, which showed high resistance to the rice blast, can be used directly in blast resistance breeding program as alternative Pib resistant sources.

Nucleotide diversity of Pita, a major blast resistance gene and identification of its minimal promoter.

Improvement of host plant resistance is one of the best methods to protect the yield from biotic stresses. Incorporation of major resistance genes or their variants into elite rice varieties will enhance the host plant resistance and its durability. Allele mining is a preferred choice to discover the novel allelic variants of major genes from wide range of germplasm. 'True' allele mining includes coding and noncoding regions, which are known to affect the plant phenotype, eventually. In this study, major blast resistance gene, Pita was analyzed by allele and promoter mining strategy and its different allelic variants were discovered from landraces and wild Oryza species. Polymorphisms at allelic sequences as well as transcription factor binding motif (TFBM) level were examined. At motif level, MYB1AT is present in Pita(Tadukan) and other resistance alleles, but was absent in the susceptible allele. Core promoter was demarked with 449 bp, employing serial promoter deletion strategy. Promoter with 1592 bp upstream region could express the gfp two fold higher than the core promoter. The identified Pita resistance allele (Pita(Konibora)) can be directly used in rice blast resistance breeding programs. Moreover, characterization of Pita core promoter led to deeper understanding of resistance gene's regulation and the identified core promoter can be utilized to express similar genes in rice.

Identification of abiotic stress miRNA transcription factor binding motifs (TFBMs) in rice.

Plant growth and yield are affected by many abiotic stresses like salinity, drought, cold and heavy metal; these stresses trigger up and down-regulate several genes through various transcription factors (TFs). Transcription factor binding motifs (TFBMs), located in the upstream region of the genes, associate with TFs to regulate the gene expression. Many factors, including the activation of miRNAs, which are encoded by genes having independent transcription units, regulate the gene expression. TFBMs in the regulatory region of miRNA sequences influence the miRNA expression, which in turn influences the expression of other genes in the cell. However, the current level of information available on TFBMs of miRNA involved in abiotic stress related defense pathway(s) is limited and in-depth studies in this direction may lead to a better understanding of their role in expression and regulation of defense responses in plants. In this study, various aspects related to genomic positions of pre-miRNA, prediction of TSS and TATA box positions and identification of known, unique motifs at regulatory regions of all the reported miRNAs of rice associated with different abiotic stresses are discussed. Sixteen motifs were identified in this study, of which nine are known cis-regulatory elements associated with various stresses, two strong motifs, (CGCCGCCG, CGGCGGCG) and five unique motifs which might play a vital role in the regulation of abiotic stresses related miRNA genes. Common motifs shared by miRNAs that are involved in more than one abiotic stresses were also identified. The motifs identified in this study will be a resource for further functional validation.

Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. The Multilaboratory Study Group.

A standardized serum bactericidal assay (SBA) is required to evaluate the functional activity of antibody produced in response to Neisseria meningitidis serogroup A and C vaccines. We evaluated assay parameters (assay buffer, target strains, growth of target cells, target cell number, complement source and concentration, and methods for growth of surviving bacteria) which may affect the reproducibility of SBA titers. The various assay parameters and specificity of anticapsular antibody to five serogroup A strains (A1, ATCC 13077, F8238, F9205, and F7485) and four serogroup C strains (C11, G7880, G8050, and 1002-90) were evaluated with Centers for Disease Control and Prevention meningococcal quality control sera. The critical assay parameters for the reproducible measurement of SBA titers were found to include the target strain, assay incubation time, and complement. The resulting standardized SBA was used by 10 laboratories to measure functional anticapsular antibody against serogroup A strains F8238 and serogroup C strain C11. In the multilaboratory study, SBA titers were measured in duplicate for 14 pairs of sera (seven adults and seven children) before and after immunization with a quadrivalent polysaccharide (A, C, Y, and W-135) vaccine. The standardized SBA was reliable in all laboratories regardless of experience in performing SBAs. For most sera, intralaboratory reproducibility was +/- 1 dilution; interlaboratory reproducibility was +/- 2 dilutions. The correlation between median titers (interlaboratory) and enzyme-linked immunosorbent assay total antibody concentrations was high for both serogroup A (r = 0.86; P < 0.001; slope = 0.5) and serogroup C (n = 0.86; P < 0.001; slope = 0.7). The specified assay, which includes the critical parameters of target strain, incubation time, and complement source, will facilitate interlaboratory comparisons of the functional antibody produced in response to current or developing serogroup A and C meningococcal vaccines.

Bactericidal antibody responses of juvenile rhesus monkeys immunized with group B Neisseria meningitidis capsular polysaccharide-protein conjugate vaccines.

Reports on the bactericidal activities of antibodies to group B Neisseria meningitidis capsular polysaccharide (B PS) are conflicting. Using three different complement sources, we analyzed the bactericidal activities of sera of juvenile rhesus monkeys immunized with five conjugate vaccines of B PS synthesized by different schemes, an Escherichia coli K92 conjugate, and a noncovalent complex of B PS with group B meningococcal outer membrane vesicles (B+OMV) (S. J. N. Devi, W. D. Zollinger, P. J. Snoy, J. Y. Tai, P. Costantini, F. Norelli, R. Rappuoli, and C. E. Frasch, Infect. Immun. 65:1045-1052, 1997). With rabbit complement, nearly all preimmune sera showed relatively high bactericidal titers, and all vaccines, except the K92 conjugate, induced a fourfold or greater increase in bactericidal titers in most of the monkeys vaccinated. In contrast, with human complement, most prevaccination sera showed no bactericidal activity and in most of the vaccine groups, little or no increase in bactericidal titer was observed. However, the covalent conjugation of P BS and OMV (B-OMV) administered with and without the Ribi adjuvant induced relatively high bactericidal titers which persisted up to 30 weeks. An analysis of the specificities of bactericidal antibodies revealed that absorption with E. coli K1 cells did not change the bactericidal titer with human complement but reduced the titers observed with the rabbit and monkey complements. A significant increase in anti-lipopolysaccharide (LPS) antibodies was elicited by the B-OMV conjugates, and nearly all of the bactericidal activity with human complement could be inhibited with the purified group B meningococcal L3,7,8 LPS. B-OMV covalently coupled via adipic acid dihydrazide elicited significantly elevated levels (P < or = 0.02) of anti-OMV antibodies compared to those of the noncovalently complexed B+OMV. An initial small-scale evaluation of B PS conjugates in adult human males appears feasible, with careful monitoring, to settle the inconsistent reports of the importance of source of complement in eliciting bacteriolysis. Subsequent analysis of resultant human antibodies for bacteriolysis, opsonophagocytosis, and protective efficacy in animal models may be the first step toward answering safety- and efficacy-related concerns about B PS conjugate vaccines.

Preclinical evaluation of group B Neisseria meningitidis and Escherichia coli K92 capsular polysaccharide-protein conjugate vaccines in juvenile rhesus monkeys.

We reported the first use of group B meningococcal conjugate vaccines in a nonhuman primate model (S. J. N. Devi, C. E. Frasch, W. Zollinger, and P. J. Snoy, p. 427-429, in J. S. Evans, S. E. Yost, M. C. J. Maiden, and I. M. Feavers, ed., Proceedings of the Ninth International Pathogenic Neisseria Conference, 1994). Three different group B Neisseria meningitidis capsular polysaccharide (B PS)-protein conjugate vaccines and an Escherichia coli K92 capsular polysaccharide-tetanus toxoid (K92-TT) conjugate vaccine are here evaluated for safety and relative immunogenicities in juvenile rhesus monkeys with or without adjuvants. Monkeys were immunized intramuscularly with either B PS-cross-reactive material 197 conjugate, B PS-outer membrane vesicle (B-OMV) conjugate, or N-propionylated B PS-outer membrane protein 3 (N-pr. B-OMP3) conjugate vaccine with or without adjuvants at weeks 0, 6, and 14. A control group of monkeys received one injection of the purified B PS alone, and another group received three injections of B PS noncovalently complexed with OMV. Antibody responses as measured by enzyme-linked immunosorbent assay varied among individual monkeys. All vaccines except B PS and the K92-TT conjugate elicited a twofold or greater increase in total B PS antibodies after one immunization. All vaccines, including the K92-TT conjugate, elicited a rise in geometric mean B PS antibody levels of ninefold or more over the preimmune levels following the third immunization. Antibodies elicited by N-pr. B-OMP3 and B-OMV conjugates were directed to the N-propionylated or to the spacer-containing B PS antigens as well as to the native B PS complexed with methylated human serum albumin. None of the vaccines caused discernible safety-related symptoms.

Binding diversity of monoclonal antibodies to alpha(2-->8) polysialic acid conjugated to outer membrane vesicle via adipic acid dihydrazide.

Murine monoclonal antibodies (mAbs) were generated using group B Neisseria meningitidis and Escherichia coli K1 polysaccharides (PSs) conjugated to outer membrane vesicle (OMV) via adipic acid dihydrazide, and were used to identify the immunodeterminants expressed on these capsular PSs. Ten mAbs representative of IgM and all subclasses of IgG were obtained which recognized diverse immunodeterminants on alpha(2-->8) polysialic acid (PSA). The specificity of mAbs to different antigenic determinants was assessed by their differential binding to PSA attached to a solid phase by different methods and confirmed by absorption studies. Two mAbs from the E. coli K1 fusion were directed to the O-acetyl epitope and the rest reacted with both the PSs only when attached to a solid phase by certain means. The methods by which PSA was coated on the solid phase had an impact on the epitope expression and binding pattern. At the concentrations used, the O-acetyl-specific mAbs, IgG1 and IgG3 mAbs were not bactericidal against group B N. meningitidis, whereas other mAbs were. The conjugates B and K1 PSs present to the murine immune system different antigenic determinants, some of which elicit bactericidal antibodies.

Preclinical efficacy of a glucuronoxylomannan-tetanus toxoid conjugate vaccine of Cryptococcus neoformans in a murine model.

The encapsulated yeast, Cryptococcus neoformans, causes life-threatening meningoencephalitis in immunocompromised humans, especially in AIDS patients. Fatality and relapse rates remain quite high despite aggressive therapy. A conjugate vaccine composed of the cryptococcal capsular glucuronoxylomannan covalently coupled to tetanus toxoid (GXM-TT) was constructed and evaluated. The vaccine elicited high levels of capsular antibodies in mice by active and passive immunizations and conferred 70-80% protection against a moderate challenge with 10(3) C, neoformans. Monitoring of serum GXM and anti-GXM antibody levels and of incidence of cryptococcal isolation from various organs of mice suggested that presence of vaccine-induced antibodies during the first 4-6 weeks of infection is critical for clearance of cryptococci from various organs, for limiting serum GXM titers from reaching immunosuppressive levels and ultimately for survival. GXM-TT is the first defined fungal vaccine to confer antibody-mediated protection against a systemic mycosis in an animal model. GXM-TT is being evaluated for safety and immunogenicity in healthy and HIV-infected human volunteers at the National Institutes of Health.

Preclinical immunoprophylactic and immunotherapeutic efficacy of antisera to capsular polysaccharide-tetanus toxoid conjugate vaccines of Vibrio vulnificus.

Vibrio vulnificus is an oyster-associated bacterial pathogen that causes life-threatening fulminating septicemia and necrotizing wound infections in humans. The capsular polysaccharide of V. vulnificus (VvPS) is critical for virulence. Previously we showed that active immunization of mice with a VvPS-tetanus toxoid (VvPS-TTa) conjugate vaccine conferred significantly higher protection against subsequent lethal challenge than immunization with VvPS alone. In the current study, we examined the utility of immunoprophylaxis or immunotherapy with hyperimmune antisera elicited by VvPS-TTa and VvPS-TTb conjugate vaccines prepared by different synthetic schemes. First we demonstrated that the Ribi adjuvant significantly enhanced the murine antibody response (P < or = 0.02) to both conjugates. Subsequently, high-titered polyclonal antisera were raised to VvPS-TTa and VvPS-TTb conjugate vaccines by using Ribi adjuvant or Freund's adjuvants. Antisera were observed to have protective effects when administered before and after acute lethal infection. All animals receiving prophylactic antisera intraperitoneally 24 h before lethal challenge with homologous carbotype 1 were protected, while 73 to 100% of control mice succumbed. Immunotherapy was also effective, with survival rates of 60 to 73% seen among mice when antisera were administered 2 h after bacterial challenge, at a time when symptoms of infection were already apparent. The protective effect of capsular antiserum appeared to be serotype specific. Antisera to the, carbotype 1 VvPS-TTa vaccine did not confer cross-protection against lethal challenge with carbotype 2 V. vulnificus despite partial structural similarity and a weak serological cross-reaction between the two carbotypes. Immune globulins induced by a potential multivalent VvPS conjugate vaccine composed of clinically prevalent carbotypes may have utility in the management of V. vulnificus infections and deserve further evaluation.

Capsular polysaccharide-protein conjugate vaccines of carbotype 1 Vibrio vulnificus: construction, immunogenicity, and protective efficacy in a murine model.

Vibrio vulnificus causes septicemia and wound infections in immunocompromised humans. The capsular polysaccharide of Vibrio vulnificus (VvPS) is critical for virulence. We synthesized conjugate vaccines of carbotype 1 VvPS under conditions and in formulations suitable for human use. Purified VvPS was conjugated to tetanus toxoid (TT) or to inactivated V. vulnificus cytolysin or elastase by two different schemes. All conjugates elicited elevated anticapsular immunoglobulin G (IgG) and IgM and antiprotein IgG responses in mice compared with saline placebo. The conjugates prepared through caboxyl activation of VvPS (VvPS-TTa, VvPS-cytolysin, and VvPS-elastase) were more immunogenic than the one prepared through hydroxyl activation (VvPS-TTb). The protective efficacy of conjugated and unconjugated formulations of VvPS and that of protein carriers were evaluated in a mouse septicemia model. Eighty percent of mice actively immunized with VvPS-TTa vaccine survived challenge with carbotype 1 V. vulnificus, while VvPS-cytolysin and VvPS-elastase conjugates conferred 44 and 40% protection, respectively. Control mice immunized with VvPS, cytolysin, or elastase alone, or saline only, showed 70 to 100% mortality. VvPS-TTa vaccine is nontoxic, immunogenic, and protective in mice.

Detection and quantitation of the glucuronoxylomannan-like polysaccharide antigen from clinical and nonclinical isolates of Trichosporon beigelii and implications for pathogenicity.

Sera from patients with systemic infections caused by the opportunistic fungus Trichosporon beigelii have been shown to cross-react with anticryptococcal antibodies. We quantitatively compared the amounts of antigen produced and examined the expression of O-acetyl epitopes from 35 strains of T. beigelii isolated from deep and superficial infections. By counterimmunoelectrophoresis, 10 of 10 isolates from deep infections were positive for polysaccharide, compared with 7 of 13 isolates from superficial infections (P = 0.02). All 23 strains tested were positive for polysaccharide when screened by immunodot. By enzyme immunoassay, the cross-reactive antigen produced by deep isolates (n = 9) had a mean titer of 1:5,500. In contrast, superficial isolates (n = 22) produced significantly less antigen than the deep isolates (P < 0.001), with a mean titer of 1:700. Isolates from environmental sources (n = 3) were similar to the superficial isolates, with a mean titer of 1:600. The mean concentrations +/- standard errors of cross-reactive polysaccharide released by deep isolates and superficial isolates were 3.09 +/- 0.44 and 1.74 +/- 0.30 micrograms/ml, respectively, when measured by rocket immunoelectrophoresis (P = 0.02). O-Acetyl epitopes were detected on polysaccharide from 8 of 9 strains of T. beigelii isolated from deep sources, while only 2 of 12 superficial isolates expressed detectable O-acetyl epitopes (P = 0.01). Thus, while all isolates of T. beigelii tested were capable of producing glucuronoxylomannan-like cross-reactive antigen, pathogenic isolates produced significantly more antigen than superficial or environmental isolates. Furthermore, significantly more pathogenic isolates than superficial or environmental isolates expressed antigen that was O acetylated.

Antibodies elicited by a Cryptococcus neoformans-tetanus toxoid conjugate vaccine have the same specificity as those elicited in infection.

The antibody responses of BALB/c mice to serotype A Cryptococcus neoformans capsular polysaccharide (CNPS) were compared after cryptococcal infection and immunization with a serotype A glucuronoxylomannan-tetanus toxoid conjugate (GXM-TT). Infection rarely resulted in a rise of serum antibody titer to CNPS. In contrast, mice immunized with GXM-TT produced serum IgM and IgG to CNPS. Six IgM and one IgG1 monoclonal antibodies (MAbs) were generated from the spleen of one infected mouse. Nine IgM, 1 IgG3, 16 IgG1, and 7 IgA MAbs were generated from the spleen of one GXM-TT-immunized mouse. All MAbs generated from both mice bound to the GXM fraction of the capsular polysaccharide. For some MAbs, de-O-acetylation of serotype A GXM abolished or greatly reduced MAb binding compared with the native GXM. All MAbs reacted with CNPS from C. neoformans serotypes A-D. MAbs generated from the infected mouse competitively inhibited the binding of MAbs generated from the GXM-TT-immunized mouse. These results indicate that some antibodies elicited by infection with C. neoformans or by immunization with GXM-TT bind to the same antigenic determinant in the GXM.

Cockroaches (Blatta and Periplaneta species) as reservoirs of drug-resistant salmonellas.

A total of 221 cockroaches (Blatta and Periplaneta spp.), collected in hospitals, houses, animal sheds, grocery stores and restaurants, in various parts of South Kanara District, a south-west coastal region of India, were studied bacteriologically for the presence of various salmonellas. Salmonellas were isolated from 4.1% of these cockroaches. Nine strains of salmonellas were recovered, belonging to five serotypes--Salmonella bovismorbificans, S. oslo, S. typhimurium, S. mbandaka and S. braenderup, the former two being the commonest serotypes. All salmonellas were resistant to one or other of 11 antibacterial drugs used in the susceptibility test. Isolation of salmonellas from cockroaches collected from the livestock premises and human dwellings suggested that they may act as significant reservoirs of salmonella in nature. Recovery of serotypes, phage types and R-types that were commonly isolated from humans and animals of this locality, suggested a transmission role for cockroaches. By harbouring potentially pathogenic, drug-resistant salmonellas, these wandering arthropods may pose dangerous infective hazards to humans and animals.

Cryptococcus neoformans serotype A glucuronoxylomannan-protein conjugate vaccines: synthesis, characterization, and immunogenicity.

We synthesized Cryptococcus neoformans serotype A glucuronoxylomannan (GXM) conjugate vaccines under conditions suitable for human use to prevent disseminated cryptococcosis. The purified, sonicated GXM was derivatized with adipic acid dihydrazide through either hydroxyl or carboxyl groups and then covalently bound to tetanus toxoid (TT) or Pseudomonas aeruginosa exoprotein A (rEPA). The immunogenicity of these conjugates was evaluated in BALB/c and general purpose mice by subcutaneous injection in saline. The conjugates elicited higher GXM antibody responses than GXM alone. Booster immunoglobulin G (IgG) and IgM responses were elicited by all conjugates in BALB/c mice. The conjugates prepared through hydroxyl activation (GXM-TT2 and GXM-rEPA) were more immunogenic than the one prepared through carboxyl activation (GXM-TT1). GXM antibody response was enhanced by the administration of monophosphoryl lipid A 2 days following the injection of GXM-TT2 (P less than 0.03). The conjugates also elicited IgG antibodies to the carrier proteins. Gel diffusion tests using conjugate-induced hyperimmune sera and chemically modified GXMs suggested that the specificity of GXM-TT1-induced antibodies was conferred by the O-acetyl groups. Hyperimmune sera generated by GXM-TT2 precipitated with the chemically unmodified and the de-O-acetylated GXMs but not with the carboxyl-reduced and de-O-acetylated GXM. GXM-TT2-induced hyperimmune serum also precipitated with the capsular polysaccharides of C. neoformans serotypes D, B, and C. The conjugate vaccines prepared through hydroxyl activation of the GXM are sufficiently immunogenic and appear to be suitable for clinical evaluation.

Antibodies to poly[(2----8)-alpha-N-acetylneuraminic acid] and poly[(2----9)-alpha-N-acetylneuraminic acid] are elicited by immunization of mice with Escherichia coli K92 conjugates: potential vaccines for groups B and C meningococci and E. coli K1.

Meningitis and other systemic infections caused by group B Neisseria meningitidis and Escherichia coli K1 remain important problems. The capsular polysaccharides (CPs) of these pathogens (poly[(2----8)-alpha-N-acetylneuraminic acid] or poly(alpha 2-8NeuNAc] are identical and are virulence factors and protective antigens for both. CP vaccines for these pathogens are not available because poly(alpha 2-8NeuNAc) alone, as a complex or a conjugate, is poorly immunogenic. Because oligomers of poly(alpha 2-8NeuNAc) in fetal brain and other tissues bind antibodies in vitro, it has been suggested that antibodies to this CP might be pathologic. We synthesized conjugates of this CP with tetanus toxoid under conditions that avoid lactone formation. Using this scheme, we also synthesized conjugates of group C meningococcal CP (poly[(2----9)-alpha-N-acetylneuraminic acid] or poly(alpha 2-9NeuNAc] and of E. coli K92 CP [poly(alpha 2-8, alpha 2-9NeuNAc)]. When injected s.c. in saline into mice, conjugates of poly(alpha 2-8NeuNAc) or poly(alpha 2-9NeuNAc) elicited homologous antibodies. E. coli K92 conjugates elicited both poly(alpha 2-8NeuNAc) and poly(alpha 2-9NeuNAc) antibodies. Both components of the conjugates expressed T-dependent immunologic properties under conditions and dosages acceptable for clinical evaluation. Poly(alpha 2-8NeuNAc) antibodies elicited by the homologous or the K92 conjugates had lower binding activities at 37 degrees C than at 22 degrees C. "Natural" poly(alpha 2-8NeuNAc) antibodies were present in almost all matched pairs of human maternal and cord sera; most cord levels were higher than in corresponding maternal sera. These findings suggest that increased levels of poly(alpha 2-8NeuNAc) IgG antibodies elicited by our conjugates will confer protective immunity to group B meningococci and E. coli K1 and will not be pathologic.