Identification of core and variable components of the Salmonella enterica subspecies I genome by microarray

We have performed microarray hybridization studies on 40 clinical isolates from 12 common serovars within Salmonella enterica subspecies I to identify the conserved chromosomal gene pool. We were able to separate the core invariant portion of the genome by a novel mathematical approach using a decision tree based on genes ranked by increasing variance. All genes within the core component were confirmed using available sequence and microarray information for S. enterica subspecies I strains. The majority of genes within the core component had conserved homologues in Escherichia coli K-12 strain MG1655. However, many genes present in the conserved set which were absent or highly divergent in K-12 had close homologues in pathogenic bacteria such as Shigella flexneri and Pseudomonas aeruginosa. Genes within previously established virulence determinants such as SPI1 to SPI5 were conserved. In addition several genes within SPI6, all of SPI9, and three fimbrial operons (fim, bcf, and stb) were conserved within all S. enterica strains included in this study. Although many phage and insertion sequence elements were missing from the core component, approximately half the pseudogenes present in S. enterica serovar Typhi were conserved. Furthermore, approximately half the genes conserved in the core set encoded hypothetical proteins. Separation of the core and variant gene sets within S. enterica subspecies I has offered fundamental biological insight into the genetic basis of phenotypic similarity and diversity across S. enterica subspecies I and shown how the core genome of these pathogens differs from the closely related E. coli K-12 laboratory strain.

Detection of mutations in Salmonella enterica gyrA, gyrB, parC and parE genes by denaturing high performance liquid chromatography (DHPLC) using standard HPLC instrumentation

Aims: Quinolone antibiotics are the agents of choice for treating systemic Salmonella infections. Resistance to quinolones is usually mediated by mutations in the DNA gyrase gene gyrA. Here we report the evaluation of standard HPLC equipment for the detection of mutations (single nucleotide polymorphisms; SNPs) in gyrA, gyrB, parC and parE by denaturing high performance liquid chromatography (DHPLC). Methods: A panel of Salmonella strains was assembled which comprised those with known different mutations in gyrA (n = 8) and fluoroquinolone-susceptible and -resistant strains (n = 50) that had not been tested for mutations in gyrA. Additionally, antibiotic-susceptible strains of serotypes other than Salmonella enterica serovar Typhimurium strains were examined for serotype-specific mutations in gyrB (n = 4), parC (n = 6) and parE (n = 1). Wild-type (WT) control DNA was prepared from Salmonella Typhimurium NCTC 74. The DNA of respective strains was amplified by PCR using Optimase (R) proofreading DNA polymerase. Duplex DNA samples were analysed using an Agilent A1100 HPLC system with a Varian Helix (TM) DNA column. Sequencing was used to validate mutations detected by DHPLC in the strains with unknown mutations. Results: Using this HPLC system, mutations in gyrA, gyrB, parC and parE were readily detected by comparison with control chromatograms. Sequencing confirmed the gyrA predicted mutations as detected by DHPLC in the unknown strains and also confirmed serotype-associated sequence changes in non-Typhimurium serotypes. Conclusions: The results demonstrated that a non-specialist standard HPLC machine fitted with a generally available column can be used to detect SNPs in gyrA, gyrB, parC and parE genes by DHPLC. Wider applications should be possible.

SPI-23 of S.Derby: role in adherence and invasion of porcine tissues

Salmonella enterica serovars Derby and Mbandaka are isolated from different groups of livestock species in the UK. S. Derby is predominantly isolated from pigs and turkeys and S. Mbandaka is predominantly isolated from cattle and chickens. Alignment of the genome sequences of two isolates of each serovar led to the discovery of a new putative Salmonella pathogenicity island, SPI-23, in the chromosome sequence of S. Derby isolates. SPI-23 is 37 kb in length and contains 42 ORFs, ten of which are putative type III effector proteins. In this study we use porcine jejunum derived cell line IPEC-J2 and in vitro organ culture of porcine jejunum and colon, to characterise the association and invasion rates of S. Derby and S. Mbandaka, and tissue tropism of S. Derby respectively. We show that S. Derby invades and associates to an IPEC-J2 monolayer in significantly greater numbers than S. Mbandaka, and that S. Derby preferentially attaches to porcine jejunum over colon explants. We also show that nine genes across SPI-23 are up-regulated to a greater degree in the jejunum compared to the colon explants. Furthermore, we constructed a mutant of the highly up-regulated, pilV-like gene, potR, and find that it produces an excess of surface pili compared to the parent strain which form a strong agglutinating phenotype interfering with association and invasion of IPEC-J2 monolayers. We suggest that potR may play a role in tissue tropism.

Identification of potential drug targets in Salmonella Typhimurium using metabolic modelling and experimental validation

almonella enterica serovar Typhimurium is an established model organism for Gram-negative, intracellular pathogens. Owing to the rapid spread of resistance to antibiotics among this group of pathogens, new approaches to identify suitable target proteins are required. Based on the genome sequence of Salmonella Typhimurium and associated databases, a genome-scale metabolic model was constructed. Output was based on an experimental determination of the biomass of Salmonella when growing in glucose minimal medium. Linear programming was used to simulate variations in energy demand, while growing in glucose minimal medium. By grouping reactions with similar flux responses, a sub-network of 34 reactions responding to this variation was identified (the catabolic core). This network was used to identify sets of one and two reactions, that when removed from the genome-scale model interfered with energy and biomass generation. 11 such sets were found to be essential for the production of biomass precursors. Experimental investigation of 7 of these showed that knock-outs of the associated genes resulted in attenuated growth for 4 pairs of reactions, while 3 single reactions were shown to be essential for growth.

Population structure and associated phenotypes of Salmonella enterica serovars Derby and Mbandaka overlap with host range

BACKGROUND:The Salmonella enterica serovar Derby is frequently isolated from pigs and turkeys whereas serovar Mbandaka is frequently isolated from cattle, chickens and animal feed in the UK. Through comparative genomics, phenomics and mutant construction we previously suggested possible mechanistic reasons why these serovars demonstrate apparently distinct host ranges. Here, we investigate the genetic and phenotypic diversity of these two serovars in the UK. We produce a phylogenetic reconstruction and perform several biochemical assays on isolates of S. Derby and S. Mbandaka acquired from sites across the UK between the years 2000 and 2010. RESULTS:We show that UK isolates of S. Mbandaka comprise of one clonal lineage which is adapted to proficient utilisation of metabolites found in soya beans under ambient conditions. We also show that this clonal lineage forms a biofilm at 25 °C, suggesting that this serovar maybe well adapted to survival ex vivo, growing in animal feed. Conversely, we show that S. Derby is made of two distinct lineages, L1 and L2. These lineages differ genotypically and phenotypically, being divided by the presence and absence of SPI-23 and the ability to more proficiently invade porcine jejunum derived cell line IPEC-J2. CONCLUSION:The results of this study lend support to the hypothesis that the differences in host ranges of S. Derby and S. Mbandaka are adaptations to pathogenesis, environmental persistence, as well as utilisation of metabolites abundant in their respective host environments.

New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin

The present study evaluated the immunogenicity of new malaria vaccine formulations based on the 19 kDa C-terminal fragment of Plasmodium vivax Merozoite Surface Protein-1 (MSP1(19)) and the Salmonella enterica serovar Typhimurium flagellin (FIiC), a Toll-like receptor 5 (TLR5) agonist. FHC was used as an adjuvant either admixed or genetically linked to the P. vivax MSP1(19) and administered to C57BL/6 mice via parenteral (s.c.) or mucosal (i.n.) routes. The recombinant fusion protein preserved MSP1(19) epitopes recognized by Sera collected from P. vivax infected humans and TLR5 agonist activity. Mice parenterally immunized with recombinant P vivax MSPI 19 in the presence of FliC, either admixed or genetically linked, elicited strong and long-lasting MSP1 (19)-specific systemic antibody responses with a prevailing IgG1 subclass response. Incorporation of another TLR agonist, CpG ODN 1826, resulted in a more balanced response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response measured by interferon-gamma secretion. Finally, we show that MSPI 19-specific antibodies recognized the native protein expressed on the surface of P. vivax parasites harvested from infected humans. The present report proposes a new class of malaria vaccine formulation based on the use of malaria antigens and the innate immunity agonist FliC. it contains intrinsic adjuvant properties and enhanced ability to induce specific humoral and cellular immune responses when administered alone or in combination with other adjuvants. (C) 2008 Elsevier Ltd. All rights reserved.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coil and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His6FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund`s adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.

CD8(+) T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (G) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS280-288 epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS280-288 peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations. (C) 2009 Elsevier Ltd. All rights reserved.

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira. (C) 2008 Elsevier Ltd. All rights reserved.

Ovine leptospirosis in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Serological investigation and PCR in detection of pathogenic leptospires in snakes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Títulos de anticorpos aglutinantes induzidos por vacinas comerciais contra leptospirose bovina

No presente estudo, 100 fêmeas bovinas foram divididas em cinco grupos de 20 animais cada. Os grupos experimentais receberam quatro diferentes vacinas comerciais (B, C, D e E), e um grupo permaneceu como controle. Amostras foram colhidas no dia da aplicação da primeira dose e nos dias 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 120, 150 e 180 pós-vacinação (PV). A triagem dos animais foi feita pela análise sorológica com 6 antígenos de leptospiras, escolhendo-se os animais não reagentes. Os títulos de anticorpos foram monitorados pela soroaglutinação microscópica (SAM) com os sorovares Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, Pomona e Wolffi. Todas as vacinas induziram, aos 3 dias PV, títulos de anticorpos aglutinantes para os sorovares Hardjo e Wolffi, que persistiram até o 150º dia PV. Os sorovares Hardjo e Wolffi induziram os maiores títulos de anticorpos aglutinantes. A vacina D, apesar de não possuir o sorovar Wolffi em sua composição foi capaz de induzir anticorpos aglutinantes contra este sorovar. Somente foram detectados anticorpos contra o sorovar Canicola nos animais vacinados com a bacterina D. A vacina que induziu os maiores títulos médios de anticorpos, considerando todos os sorovares testados foi a D.

Nose-to-nose transmission of Salmonella Typhimurium between weaned pigs

Little attention has been paid to the possibility of transmission of Salmonella in intensive pig production systems through alternate methods, such as airborne or direct nose-to-nose contact. This experimental study tested the hypothesis of nose-to-nose transmission of Salmonella enterica serovars Typhimurium (Trial I) and Agona (Trial II) in weaned pigs using stainless steel/ glass isolation cabinets. In each trial, cabinet 1 (control pigs) and cabinet 2 (sentinel pigs) were connected directly to the fan unit. Cabinet 3 (seeded pigs) was not directly linked to the fan, but was arranged to receive a constant unidirectional airflow from cabinet 2 (sentinel pigs) through a 10 cm diameter hole, which also allowed nose-to-nose contact between pigs housed in these two cabinets. Air was taken out of the system through ducts connecting cabinets 1 and 3 to the exhauster. Therefore, direct contact among seeded and sentinel pigs was allowed but possible aerial transference of contaminated particles between those cabinets was prevented. The system was opened 21 days post-inoculation and tissue samples were collected for bacteriological analysis. The recovery of nalidixic acid-resistant Salmonella Typhimurium from sentinel pigs corroborates the hypothesis of nose-to-nose transmission of that pathogen in pigs. However, serovar-related differences might exist regarding the nose-to-nose transmissibility of Salmonella in pigs, since Salmonella Agona was not detected in sentinel pigs (Trial II). Published by Elsevier B.V.

Serosurvey for antibodies against Brucella abortus and Leptospira interrogans in pampas deer from Brazil

A survey for antibodies against Brucella abortus, and Leptospira interrogans was conducted on 17 pampas deer (Ozotocerus bezoarticus) from Pantanal Matogrossense (State of Mato Grosso do Sul, Brazil) and on 24 pampas deer from Parque Nacional de Emas (State of Goias, Brazil). Antibodies against B. abortus were detected by plate ag glutination, rose Bengal, and complement fixation tests, antibodies against Leptospira interrogans were detected by the microscopic agglutination test. All sera were negative for B. abortus antibodies and all deer sera from Parque Nacional de Emas were negative for L. interrogans antibodies. Four (24%) of 17 sera from Pantanal Matogrossense were positive for L. interrogans serovar (n = 2) hardjo, wolffi (n = 1) and mini (n = 1). While these diseases do not appear to be of major importance to the health status of Pampas deer, it appears that deer are reservoir for leptospirosis in one of the study areas.

Evaluation of the counterimmunoelectrophoresis reaction as a serodiagnosis test for swine leptospirosis

Avaliou-se a reação de contraimunoeletroforese (CIE) como teste gênero-específico para diagnóstico da leptospirose suína, usando-se três extratos solúveis de Leptospira sp, sorovares pomona, icterohaemorrhagiae e patoc, obtidos pelo tratamento com Triton X-100 a quente e aplicados a amostras de soro de suínos subdivididos em três grupos: Grupo 1, 10 suínos experimentalmente infectados com estirpe Pomona; Grupo 2, 50 suínos naturalmente infectados e Grupo 3, controle. As amostras de soros foram submetidas à reação de CIE e os resultados comparados aos da Soroaglutinação Microscópica (SAM), técnica de referência pela WHO. Os Grupos 1 e 3 foram monitorados por 93 dias após a inoculação (p.i.). Pela SAM a soroconversão do Grupo 1 ocorreu por volta do 10º dia p.i., enquanto pela CIE, empregando-se qualquer extrato antigênico, foi anterior à SAM. Quando a CIE foi realizada frente a antigeno homólogo à infecção, seus resultados foram equivalentes aos da SAM, não se verificando o mesmo frente aos antígenos heterólogos. Neste aspecto, os Grupos 1 e 3 mostraram comportamento diferente pois não houve diferença significativa entre os resultados da CIE frente aos três antígenos, o que poderia significar serem independentes do sorovar responsável pelo surto ou infectante. Embora a CIE seja segura, rápida, de fácil execução, de baixo custo e ideal para análise em grande escala de amostras, revelou-se de limitada capacidade gênero-específica, o que não é desejavel para testes de triagem de campo; mas poderia ser útil na detecção precoce de resposta sorológica em relação à SAM.