Monoclonal antibody of IgG isotype against a cross-reactive lipopolysaccharide epitope of Chlamydia and Salmonella Re chemotype enhances infectivity in L-929 fibroblast cells

A murine monoclonal antibody (MAb) 202D7 of IgG3 isotype recognizes a lipopolysaccharide (LPS) epitope of Chlamydia spp. and cross-reacts with the Re chemotype LPS of Salmonella and Escherichia coli. The antibody exhibits strong complement activating properties and stimulates phagocytosis of Salmonella enterica serovar Minnesota Re mutant by murine macrophages. Salmonella Re mutants are non-invasive for cell monolayers but still can enter and replicate in L-929 murine fibroblast cells. The entry of bacteria within the cells increases five-fold in the presence of MAb 202D7. The antibody mediates attachment and enhances five-fold the infectivity of Chlamydia pneumoniae into L-929 cells, which suggests a possible IgG-mediated mechanism of entry and survival of the pathogen in fibroblast cells.     

Conformational epitope mapping of OmpC, a major cell surface antigen from Salmonella typhi

The outer membrane protein OmpC, a trimer made of 16 stranded beta-barrel monomers, is a major cell surface antigen from the human pathogen Salmonella typhi. The relative stability of the epitopes recognising a Salmonella specific MAb (referred as MPN5) and an Enterobacteria specific MAb (referred as P7D8) and the role of the trimeric organisation has been probed using gel electrophoresis and monoclonal antibodies. The assembly of the trimer and the stability of the beta-barrel are found to be important for epitope presentation. The Salmonella specific conformational epitope is found to be more stable than the Enterobacteria specific one. The important residues of the Salmonella specific (Asp 25 of loop 1, Asp 340 of loop 8, Lys 334 of loop 8, and Tyr 210 of loop 5) and the Enterobacteria specific (Asp 25 of loop 1, Tyr 210 of loop 5, and Lys 152 of loop 4) conformational epitope have been identified using monoclonal antibodies, chemical modification, and solid phase binding methods.     

Use of Polymerase Chain Reaction and Restriction Enzyme Analysis to directly detect and identify Salmonella typhimurium in food

A primer set of oligonucleotides (Salm 3 and Salm 4) from the inv A gene of Salmonellae has been evaluated for the specific detection of Salmonella spp. by the polymerase chain reaction (PCR). This primer set amplified 33 Salmonella serovars but did not amplify 16 non- Salmonella bacteria. Moreover, after PCR amplification, it was possible to identify Salm. typhimurium by restriction enzyme analysis. The PCR-RE method developed could represent a helpful tool for detecting Salmonella spp., and for directly and rapidly identifying Salm. typhimurium in food.     

Monoclonal antibody specific for Listeria monocytogenes associated with a 66-kilodalton cell surface antigen.

A monoclonal antibody (MAb), EM-7G1, specific for Listeria monocytogenes was developed by using a previously developed MAb, C11E9 (A. K. Bhunia, P. H. Ball, A. T. Fuad, B. W. Kurz, J. W. Emerson, and M. G. Johnson, Infect. Immun. 59:3176-3184, 1991), to mask epitopes shared by L. monocytogenes and Listeria innocua in a 66-kDa cell surface protein. MAb EM-7G1 was an immunoglobulin subclass G1 antibody with kappa light chains. This MAb reacted with all 34 strains of L. monocytogenes tested and showed no cross-reaction with other Listeria spp. or other gram-positive or gram-negative organisms tested by enzyme-linked immunosorbent assay, dot blotting, and colony blotting. A second MAb, EM-6E11, reacted with all Listeria spp. tested but no other bacteria. In a Western blot (immunoblot) assay, EM-7G1 reacted with a crude cell surface protein of 66 kDa with a pI value of 6.7, while EM-6E11 reacted with two protein bands of 43 and 94 to 97 kDa with pI values of 4.0 and 4.3, respectively. Results with trypsin or pronase treatments indicated that the cell antigen reacting with EM-7G1 was on the surface of L. monocytogenes V7 and Scott A cells.     

Use of two 16S DNA targeted oligonucleotides as PCR primers for the specific detection of Salmonella in foods

A 16S DNA targeted polymerase chain reaction (PCR) method specific for the detection of Salmonella isolates with various serotypes was developed. The primers used for such a PCR method were 16SF1 and 16SIII. 16SF1 is the reverse and complementary strand of 16SI which has been shown to be able to hybridize with Salmonella and Citrobacter spp. 16III on the other hand, is able to hybridize with Klebsiella and Serratia spp. in addition to Salmonella. Although 16SF1 and 16SIII were not specific to Salmonella only, when they were used as PCR primers, only the Salmonella isolates could be specifically detected. The interference from Citrobacter, Klebsiella and Serratia spp. could be prevented. None of the other non- Salmonella isolates including strains of the family of Enterobacteriaceae closely related to Salmonella would generate the false-positive reaction. When this PCR system was used for the detection of Salmonella cells artificially contaminated in food samples, results obtained were satisfactory. A detection limit of N × 10⁰ cells per assay could be obtained.     

Flagella and Motility in Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae has been considered nonmotile and nonflagellate. In this work, it is demonstrated that A. pleuropneumoniae produces flagella composed of a 65-kDa protein with an N-terminal amino acid sequence that shows 100% identity with those of Escherichia coli, Salmonella, and Shigella flagellins. The DNA sequence obtained through PCR of the fliC gene in A. pleuropneumoniae showed considerable identity (93%) in its 5' and 3' ends with the DNA sequences of corresponding genes in E. coli, Salmonella enterica, and Shigella spp. The motility of A. pleuropneumoniae was observed in tryptic soy or brain heart infusion soft agar media, and it is influenced by temperature. Flagella and motility may be involved in the survival and pathogenesis of A. pleuropneumoniae in pigs.     

New plate medium for facilitated differentiation of Salmonella spp. from Proteus spp. and other enteric bacteria

A new agar medium for the differentiation of Salmonella spp. from other members of the family Enterobacteriaceae is described. This medium exploits a novel phenotypic characteristic of Salmonella spp.: the formation of acid from propylene glycol. This characteristic may be used in combination with a chromogenic indicator of beta-galactosidase to differentiate Salmonella spp. from Proteus spp. and the other members of the Enterobacteriaceae. Desoxycholate may be included in the plate medium as an inhibitor of gram-positive organisms. Non-typhi Salmonella spp. yield distinct, bright red colonies on this medium, allowing facilitated identification and unambiguous differentiation from Proteus spp.     

New plate medium for facilitated differentiation of Salmonella spp. from Proteus spp. and other enteric bacteria.

A new agar medium for the differentiation of Salmonella spp. from other members of the family Enterobacteriaceae is described. This medium exploits a novel phenotypic characteristic of Salmonella spp.: the formation of acid from propylene glycol. This characteristic may be used in combination with a chromogenic indicator of beta-galactosidase to differentiate Salmonella spp. from Proteus spp. and the other members of the Enterobacteriaceae. Desoxycholate may be included in the plate medium as an inhibitor of gram-positive organisms. Non-typhi Salmonella spp. yield distinct, bright red colonies on this medium, allowing facilitated identification and unambiguous differentiation from Proteus spp.     

Transfer of the Salmonella type III effector sopE between unrelated phage families.

Salmonella spp. are pathogenic enterobacteria that employ type III secretion systems to translocate effector proteins and modulate responses of host cells. The repertoire of translocated effector proteins is thought to define host specificity and epidemic virulence, and varies even between closely related Salmonella strains. Therefore, horizontal transfer of effector protein genes between Salmonella strains plays a key role in shaping the Salmonella-host interaction. Several effector protein genes are located in temperate phages. The P2-like phage SopE Phi encodes SopE and the lambda-like GIFSY phages encode several effector proteins of the YopM/IpaH-family. Lysogenic conversion with these phages is responsible for much of the diversity of the effector protein repertoires observed among Salmonella spp. However, free exchange of effector proteins by lysogenic conversion can be restricted by superinfection immunity. To identify genetic mechanisms that may further enhance horizontal transfer of effector genes, we have analyzed sopE loci from Salmonella spp. that do not harbor P2-like sequences of SopE Phi. In two novel sopE loci that were identified, the 723 nt sopE gene is located in a conserved 1.2 kb cassette present also in SopE Phi. Most strikingly, in Salmonella enterica subspecies I serovars Gallinarum, Enteritidis, Hadar and Dublin, the sopE-cassette is located in a cryptic lambda-like prophage with similarity to the GIFSY phages. This provides the first evidence for transfer of virulence genes between different phage families. We show that such a mechanism can circumvent restrictions to phage-mediated gene transfer and thereby enhances reassortment of the effector protein repertoires in Salmonella spp.     

Two murine monoclonal antibodies against serogroup E Salmonellae

A monoclonal antibody (MAb), MO15, was raised against the lipopolysaccharide antigen of an epsilon15-lysogenized serogroup E(1) Salmonella strain. The O factor 15-specific MAb MO15, together with another serogroup E-specific MAb, can differentiate among phage lysogenization variants in serogroup E salmonellae. Their epitope specificities in relation to conventional O-antigenic structures are discussed.     

Survey of co-infection by Salmonella and oxyurids in tortoises

ABSTRACT: BACKGROUND: Salmonella spp. and oxyurids are among the most prevalent bacterial and parasitic agents in reptiles. These organisms are routinely isolated in healthy tortoises, although heavy infections may cause significant pathology. Tortoises are considered a common source of reptileassociated salmonellosis, an important zoonosis reported worldwide. A survey of the prevalence of almonella spp. and oxyurids in 53 tortoises was conducted in southern Italy and a possible correlation between the two pathogens was therefore investigated. RESULTS: Salmonella spp. and oxyurids were detected with a prevalence of 49.1 and 81.1%, respectively. A significant positive correlation between Salmonella spp. and oxyurids was demonstrated. However, confounding factors related to husbandry could have been involved in determining this correlation. CONCLUSIONS: Our results suggest that caution should be exercised in translocation, husbandry, and human contact with tortoises and other exotic pets. Further studies on the epidemiology, molecular characterization and pathogenesis of Salmonella and oxyurids are needed to assess the actual impact of these organisms, as single or associated infections, on tortoises and on other exotic pets.     

Identification and differentiation of Panax species using ELISA, RAPD and eastern blotting

Immunochemical and genetic methods have been developed in order to distinguish Panax spp. With the aim of establishing immunochemical methods, two hybridomas (3H4 and 5H8), each secreting a monoclonal antibody (MAb) against proteins of Panax ginseng, were prepared by fusing splenocytes immunized with two kinds of ginseng water-soluble fractions and a hypoxanthine-thymidine-aminopterin-sensitive mouse myeloma cell line, P3-X63-Ag8-U1. MAb 3H4 cross-reacted with four Panax spp., whereas the MAb 5H8 cross-reacted with P. ginseng in the enzyme-linked immunosorbent assay (ELISA). ELISA and western blotting methods using a ginseng water-soluble fraction as the solid-phase antigen were developed for the unambiguous authentication of P. ginseng. A combination of random amplified polymorphic DNA (RAPD) and eastern blotting analyses using anti-ginsenoside Rb1 and Rgl monoclonal antibodies was used for the identification of P. notoginseng, P. quinquefolius and P. japonicus. RAPD can be used to differentiate the species of Panax from each other. An important parameter used for differentiating P. notoginseng is the absence of ginsenoside Rc in the extract of P. notoginseng with eastern blotting. The combination of these methods enabled a reliable identification of Panax spp.     

Salmonella host cell invasion emerged by acquisition of a mosaic of separate genetic elements, including Salmonella pathogenicity island 1 (SPI1), SPI5, and sopE2

Salmonella spp. possess a conserved type III secretion system encoded within the pathogenicity island 1 (SPI1; centisome 63), which mediates translocation of effector proteins into the host cell cytosol to trigger responses such as bacterial internalization. Several translocated effector proteins are encoded in other regions of the Salmonella chromosome. It remains unclear how this complex chromosomal arrangement of genes for the type III apparatus and the effector proteins emerged and how the different effector proteins cooperate to mediate virulence. By Southern blotting, PCR, and phylogenetic analyses of highly diverse Salmonella spp., we show here that effector protein genes located in the core of SPI1 are present in all Salmonella lineages. Surprisingly, the same holds true for several effector protein genes located in distant regions of the Salmonella chromosome, namely, sopB (SPI5, centisome 20), sopD (centisome 64), and sopE2 (centisomes 40 to 42). Our data demonstrate that sopB, sopD, and sopE2, along with SPI1, were already present in the last common ancestor of all contemporary Salmonella spp. Analysis of Salmonella mutants revealed that host cell invasion is mediated by SopB, SopE2, and, in the case of Salmonella enterica serovar Typhimurium SL1344, by SopE: a sopB sopE sopE2-deficient triple mutant was incapable of inducing membrane ruffling and was >100-fold attenuated in host cell invasion. We conclude that host cell invasion emerged early during evolution by acquisition of a mosaic of genetic elements (SPI1 itself, SPI5 [sopB], and sopE2) and that the last common ancestor of all contemporary Salmonella spp. was probably already invasive.     

Development of liposome immunoassay for salmonella spp. using immunomagnetic separation and immunoliposome

The ability to detect Salmonella spp. is essential in the prevention of foodborne illness. This study examined a Salmonella spp. detection method involving the application of immunomagnetic separation and immunoliposomes (IMS/IL) encapsulating sulforhodamine B (SRB), a fluorescent dye. A quantitative assay was conducted by measuring the fluorescence intensity of SRB that was produced from an immunomagnetic bead-Salmonella spp.-immunoliposome complex. The results indicated detection limits of 2.7x10(5) and 5.2x10(3) CFU/ml for Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), respectivley. The signal/noise ratio was improved by using 4% skim milk as a wash solution rather than 2% BSA. In addition, higher fluorescence intensity was obtained by increasing the liposome size. Compared with the conventional plating method, which takes 3-4 days for the isolation and identification of Salmonella spp., the total assay time of 10 h only including 6 h of culture enrichment was necessary for the Salmonella detection by IMS/IL. These results indicate that the IMS/ IL has great potential as an alternative rapid method for Salmonella detection.     

Campylobacteriosis, salmonellosis, and shigellosis in free-ranging human-habituated mountain gorillas of Uganda

For conservation purposes and due to growing ecotourism, free-ranging mountain gorillas (Gorilla gorilla beringei) have been habituated to humans. Fecal specimens (n = 62) collected in January 1999 from mountain gorillas of the Bwindi and Mgahinga National Parks, Uganda, were tested for Campylobacter spp., Salmonella spp., and Shigella spp., and the overall prevalence of infection was 19%, 13%, and 6%, respectively. The prevalence of positive specimens was not related to the year of habituation of a gorilla group to humans. Campylobacter spp., Salmonella, and Shigella spp. infections were not distributed equally among the age classes of gorillas; most of the enteropathogens (80%), and all Shigella spp. organisms, S. sonnei, S. boydii, and S. flexneri, were isolated from subadults and adult gorillas with ages ranging from 6.0 to 11.9 yr. The prevalence of Campylobacter spp. and Salmonella spp. infections among human-habituated gorillas has doubled during the last 4 yr, and isolation of Shigella spp. for the first time from mountain gorillas, may indicate enhanced anthropozoonotic transmission of these enteropathogens.     

Survival of Salmonella spp. in a simulated acid-phase anaerobic digester treating sewage sludge

The presence of pathogenic microorganisms in municipal waste sludge may create a serious outbreak of water borne diseases if the sludge is used for agricultural purpose. An attempt to decrease the number of pathogenic microorganisms, Salmonella spp. using a simulated acid-phase anaerobic digester was tested in a laboratory-scale batch experiment. Reduction of Salmonella spp. was demonstrated in a mixture of sludge and organic acid, simulating an acid digester of a two-phase anaerobic digestion process. A high concentration of organic acid at a pH value of 5.5-6.0 prevents a decrease in Salmonella spp. concentration. Almost complete destruction of Salmonella spp. is observed within two days if the pH value is maintained below 5.5.     

Survival of Salmonella spp. in a simulated acid-phase anaerobic digester treating sewage sludge

The presence of pathogenic microorganisms in municipal waste sludge may create a serious outbreak of water borne diseases if the sludge is used for agricultural purpose. An attempt to decrease the number of pathogenic microorganisms, Salmonella spp. using a simulated acid-phase anaerobic digester was tested in a laboratory-scale batch experiment. Reduction of Salmonella spp. was demonstrated in a mixture of sludge and organic acid, simulating an acid digester of a two-phase anaerobic digestion process. A high concentration of organic acid at a pH value of 5.5-6.0 prevents a decrease in Salmonella spp. concentration. Almost complete destruction of Salmonella spp. is observed within two days if the pH value is maintained below 5.5.     

Genetic analysis of multiple antimicrobial resistance in Salmonella isolated from diseased broilers in Egypt

To date, no information has been available on the molecular bases of antimicrobial resistance in Salmonella spp. from poultry in Egypt or even in Africa. Therefore, the objective of this study was to analyze, at the molecular level, the mechanisms of multidrug-resistance in isolates of Salmonella recovered from diseased broilers in Egypt. Twenty-one Salmonella isolates were identified; 13 of these isolates were Salmonella enterica serovar Enteritidis and eight Salmonella enterica serovar Typhimurium. 17 (81%). Salmonella isolates displayed multidrug resistance phenotypes, particularly against ampicillin, streptomycin, spectinomycin, kanamycin, tetracycline, chloramphenicol, and trimethoprim/sulfamethoxazole. PCR and DNA sequencing identified class 1 integrons in nine (42.9%) isolates and class 2 integrons in three (14.3%) isolates. The identified resistance genes within class 1 integrons were aminoglycoside adenyltransferase type A, aadA1, aadA2 and aadA5 and dihydrofolate reductase type A, dfrA1, dfrA5, dfrA12, dfrA15 and dfrA17. The β-lactamase encoding genes bla(TEM-1) and bla(CMY-2) and florfenicol resistance gene floR were also identified. Furthermore, the tetracycline resistance gene tet(A) was identified in 14 (66.7%) Salmonella isolates. To the best of our knowledge, this is the first report of the molecular basis of antimicrobial resistance in Salmonella spp. isolated from poultry in Africa.     

Climate patterns governing the presence and permanence of salmonellae in coastal areas of Bahia de Todos Santos, Mexico

Despite the importance of salmonellae as one of the major causes of food-borne infections worldwide, data regarding the presence of these organisms in the environment are limited. We investigated the presence of Salmonella spp. in Bahia de Todos Santos (Baja California, Mexico) and evaluated the environmental factors that affect the occurrence of Salmonella spp. in this arid region. A total of 1,331 samples collected from 21 sites along the coast during a period of 3 years were analyzed for Salmonella spp. Geographical and seasonal distribution of Salmonella spp. was evaluated in association with environmental parameters and with human infections in the area. The incidence of Salmonella bacteria throughout the study was 4.8%, with the highest incidence detected in wastewater (16.2%), followed by stream water (10.6%), mollusks (7.4%), and seawater (2.3%). Twenty different serotypes were identified among the 64 Salmonella isolates. The dominant serotype was Typhimurium (23.4%), followed by Vejle (6.2%). The presence of Salmonella spp. in coastal areas was mostly confined to rainy periods and areas of stream discharges, and runoff was identified as the predominant factor influencing the transport of Salmonella bacteria from source points to the sea via streams. Isolation of Salmonella spp. was negatively and significantly associated with temperature, probably because of the effect of solar radiation in the decline of permanence of Salmonella bacteria. Conversely, human infections prevailed during the warmest months and were negatively correlated with the presence of Salmonella spp. in the marine environment.     

Airborne microorganisms in a municipal solid waste recovery system

The types of bacteria and fungi present in the air of a municipal solid waste recovery system have been characterized and the population densities estimated. Conventional methods were successful in enumerating coagulase-positive staphylococci, Klebsiella spp., gram-negative bacteria, fungi, and common indicator organisms. Selective and enrichment media, however, did not yield Salmonella isolates. Salmonella and Shigella were recovered at a frequency of 3% or less on trypticase soy agar. A broad spectrum of bacteria and fungi were isolated. No evidence has been found that indicates that these organisms have produced adverse health effects.