A novel fimbrial haemagglutinin produced by a strain of Salmonella of serotype Salinatis

A strain of Salmonella of serotype Salinatis, that produced a mannose-resistant and eluting haemagglutinin (MREHA) when cultured at 37 degrees C but not at 18 degrees C, was examined by electron microscopy after negative staining. Production of this MREHA, previously described as being non-fimbrial, was correlated with the presence of thin fimbriae which had an external diameter of 3.6 nm. The purified Salinatis thin fimbriae had an estimated Mr of 19 kDa. This fimbrial MREHA was not produced by strains of the antigenically related serotypes Duisburg and Sandiego.     

A high-molecular-weight outer membrane protein of Xanthomonas oryzae pv. oryzae exhibits similarity to non-fimbrial adhesins of animal pathogenic bacteria and is required for optimum virulence

Transposon insertions in a novel 3.798 kb open reading frame (ORF) of the rice pathogen, Xanthomonas oryzae pv. oryzae (Xoo) cause virulence deficiency and altered colony/lawn morphology. This ORF encodes a protein, XadA, of 1,265 amino acids that exhibits significant similarity to non-fimbrial adhesins of animal pathogenic bacteria such as Yersinia YadA and Moraxella UspA1. An interesting feature is that the YadA similarity region is repeated six times within the XadA sequence and encompasses almost the entire length of the protein. Anti-XadA antibodies identified a 110 kDa outer membrane protein that was sensitive to protease treatment of whole cells. XadA expression is induced in minimal medium. Homology modelling suggests that XadA adopts a beta-helix conformation-like pertactin, a non-fimbrial adhesin of Bordetella pertussis. This work is the first characterization of a non-fimbrial adhesin-like molecule in a plant pathogenic bacterium. It extends our knowledge about the repertoire of homologous virulence factors that are deployed by animal and plant pathogenic bacteria to include functions potentially involved in adhesion.     

Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins

The non-fimbrial adhesins, YadA of enteropathogenic YERSINIA: species, and UspA1 and UspA2 of Moraxella catarrhalis, are established pathogenicity factors. In electron micrographs, both surface proteins appear as distinct 'lollipop'-shaped structures forming a novel type of surface projection on the outer membranes. These structures, amino acid sequence analysis of these molecules and yadA gene manipulation suggest a tripartite organization: an N-terminal oval head domain is followed by a putative coiled-coil rod and terminated by a C-terminal membrane anchor domain. In YadA, the head domain is involved in autoagglutination and binding to host cells and collagen. Analysis of the coiled-coil segment of YadA revealed unusual pentadecad repeats with a periodicity of 3.75, which differs significantly from the 3.5 periodicity found in the Moraxella UspAs and other canonical coiled coils. These findings predict that the surface projections are formed by oligomers containing right- (Yersinia) or left-handed (Moraxella) coiled coils. Strikingly, sequence comparison revealed that related proteins are found in many proteobacteria, both human pathogenic and environmental species, suggesting a common role in adaptation to specific ecological niches.     

A new mannose-resistant haemagglutinin in Klebsiella

Strains of Klebsiella of the species (or 'patho-bio-sero-geogroups') Kl. atlantae, Kl. edwardsii and Kl. rhinoscleromatis produced neither haemagglutinins (HAs) nor fimbriae; strains of Kl. ozaenae were HA- but some produced type-6 fimbriae; and strains of Kl. pneumoniae (sensu stricto) and Kl. aerogenes that produced the mannose-sensitive HA (MS-HA) formed type-1 fimbriae. Most strains of Kl. aerogenes produced, in addition, one or both of the mannose-resistant HAs, MR/K-HA or MR/P-HA. The former, associated with type-3 fimbriae, was produced by 95%, and the latter by 57%, of the Kl. aerogenes strains. Some of the properties of the MR/P-HA, apparently a non-fimbrial HA not previously recognised in Klebsiella, are described.     

Adhesins of escherichia coli

E. coli has got increasing importance as a causative agent of intestinal and extra-intestinal diseases. In both these infections adhesion of the bacteria to mucous surface cells are initial events for coionization and development of infection. Adhesins are bacterial recognition proteins which specifically interact with carbohydrate moieties of glycoproteins or glycolipids on mammalian cells. The adhesiveness of bacteria is associated with filamentous surface appendages, designated as fimbriae or pili, as well as with non-fimbrial components. Some recent data on the nomenclature, classification, disease association, receptor specificity, and topographic arrangement are presented. The correlation between E. coli O : K : H serovar and fimbrial antigens is demonstrated on the basis of E. coli isolated from patients with urinary tract infections. Hitherto unknown non-fimbrial adhesins are briefly described.     

A new mannose-resistant haemagglutinin in Klebsiella

Strains of Klebsiella of the species (or 'patho-bio-sero-geogroups') Kl. atlantae, Kl. edwardsii and Kl. rhinoscleromatis produced neither haemagglutinins (HAs) nor fimbriae; strains of Kl. ozaenae were HA^- but some produced type-6 fimbriae; and strains of Kl. pneumoniae ( sensu stricto ) and Kl. aerogenes that produced the mannose-sensitive HA (MS-HA) formed type-1 fimbriae. Most strains of Kl. aerogenes produced, in addition, one or both of the mannose-resistant HAs, MR/K-HA or MR/P-HA. The former, associated with type-3 fimbriae, was produced by 95%, and the latter by 57%, of the Kl. aerogenes strains. Some of the properties of the MR/P-HA, apparently a non-fimbrial HA not previously recognised in Klebsiella , are described.     

Genetic analysis of the O-antigen gene clusters of Yersinia pseudotuberculosis O:6 and O:7

Among the 21 O-polysaccharide (OPS) O-antigen-based serotypes described for Yersinia pseudotuberculosis, those of O:6 and O:7 are unusual in that both contain colitose (4-keto-3,6-dideoxy-d-mannose or 4-keto-3,6-dideoxy-l-xylo-hexose), which has not otherwise been reported for this species, and the O:6 OPS also contains yersiniose A (4-C[(R)-1-hydroxyethyl]-3,6-dideoxy-d-xylo-hexose), another unusual dideoxyhexose sugar. In Y. pseudotuberculosis, the genes for OPS synthesis generally cluster together between the hemH and gsk loci. Here, we present the sequences of the OPS gene clusters of Y. pseudotuberculosis O:6 and O:7, and the location of the genes required for synthesis of these OPSs, except that there is still ambiguity regarding allocation of some of the glycosyltransferase functions. The O:6 and O:7 gene clusters have much in common with each other, but differ substantially from the group of 13 gene clusters already sequenced, which share several features and sequence similarities. We also present a possible sequence of events for the derivation of the O:6 and O:7 gene clusters from the most closely related set of 13 sequenced previously.     

Transmission of Yersinia enterocolitica 4/O:3 to pets via contaminated pork

AIMS: This study was conducted to investigate sources of Yersinia enterocolitica 4/O:3 infections in dogs and cats. METHODS AND RESULTS: Transmission of Y. enterocolitica 4/O:3 to pets via contaminated pork was studied using PFGE with NotI, ApaI and XhoI enzymes. A total of 132 isolates, of which 16 were from cat and dog faeces and 116 from raw pork samples, were recovered in Finland during 1998-99. Cat 1, whose diet consisted mostly of raw pig hearts and kidneys, excreted Y. enterocolitica 4/O:3 of genotype G4. This predominant genotype was also found in isolates recovered from the pig heart, liver, kidney, tongue and ear, and minced pork samples. Dog 2, which was fed raw minced pork, excreted Y. enterocolitica of genotype G13. This genotype was also identified in isolates recovered from the pig heart, kidney and tongue, and minced pork samples. CONCLUSION: These results show that raw pork can be an important source of Yersinia enterocolitica 4/O:3 infections in dogs and cats. Significance and Impact of the Study: Raw pork should not be given to pets.     

Alternative localization of the determinant of pathogenicity coded by the Yersinia pseudotuberculosis pVM82 plasmid]

The chromosomal DNA regions in Yersinia pseudotuberculosis strains occur that are homologous to 25 Md DNA segment of the plasmid pVM82 encoding the bacterial capability of immunosuppression. The character of the chromosomal DNA regions dispersion reacting with the 25 Md segment probes is different in epidemiologically hazardous and nonvirulent strains of Yersinia pseudotuberculosis. The specific DNA regions occur as well as identical ones. The suppression of antibody formation to a number of main Yersinia pseudotuberculosis antigens by epidemiologically hazardous strain is demonstrated. The suppression is analogous to the one previously described for Yersinia pseudotuberculosis strains harbouring the plasmid pVM82.     

Extracts of protozoa contain materials that react specifically in the immunoassay for guinea pig insulin

Extracts of protozoa contain materials that resemble guinea pig insulin, which is noted for its unusual structure and properties. The protozoan derived materials react in the radioimmunoassay for guinea pig insulin; some but not all of these immunoreactive materials migrate on gel filtration in the position of authentic guinea pig insulin. Experiments were done to exclude artifacts in the assay as well as inadvertent contamination by guinea pig insulin. By immunological methods, we segregated the guinea pig type immunoactivity from that which has rat/pork type immunoactivity. These findings extend our studies of extracts of guinea pig tissues which also have these two types of insulin immunoactivities.     

Comparative chromosome painting defines the high rate of karyotype changes between pigs and bovids

Human and sheep chromosome-specific probes were used to construct comparative painting maps between the pig (Suiformes), cattle and sheep (Bovidae), and humans. Various yet unknown translocations were observed that would assist in a more complete reconstruction of homology maps of these species. The number of homologous segments that can be identified with sheep probes in the pig karyotype exceeds that described previously by chromosome painting between two non-primate mammals belonging to the same order. Sheep probes painted 62 segments on pig autosomes and delineated not only translocations, but also 9 inversions. All inversions were paracentric and indicate that these rearrangements may be characteristic for chromosomal changes in suiforms. Hybridizations of all sheep painting probes to cattle chromosomes confirmed the chromosome conservation in bovids. In addition, we observed a small translocation that was previously postulated from linkage mapping data, but was not yet described by physical mapping. The chromosome painting data are complemented with a map of available comparative gene mapping data between pig and sheep genomes. A detailed table listing the comparative gene mapping data between pig and cattle genomes is provided. The reanalysis of the pig karyotype with a new generation of human paint probes provides an update of the human/pig comparative genome map and demonstrates two new chromosome homologies. Seven conserved segments not yet identified by chromosome painting are also reported. Received: 2 October 2000 / Accepted: 15 January 2001     

Natural Pig Plasma Immunoglobulins Have Anti-Bacterial Effects: Potential for Use as Feed Supplement for Treatment of Intestinal Infections in Pigs

There is an increasing demand for non-antibiotics solutions to control infectious disease in intensive pig production. Here, one such alternative, namely pig antibodies purified from slaughterhouse blood was investigated in order to elucidate its potential usability to control post-weaning diarrhoea (PWD), which is one of the top indications for antibiotics usage in the pig production. A very cost-efficient and rapid one-step expanded bed adsorption (EBA) chromatography procedure was used to purify pig immunoglobulin G from slaughterhouse pig plasma (more than 100 litres), resulting in >85% pure pig IgG (ppIgG). The ppIgG thus comprised natural pig immunoglobulins and was subsequently shown to contain activity towards four pig-relevant bacterial strains (three different types of Escherichia coli and one type of Salmonella enterica) but not towards a fish pathogen (Yersinia ruckeri), and was demonstrated to inhibit the binding of the four pig relevant bacteria to a pig intestinal cell line (IPEC-J2). Finally it was demonstrated in an in vivo weaning piglet model for intestinal colonization with an E. coli F4+ challenge strain that ppIgG given in the feed significantly reduced shedding of the challenge strain, reduced the proportion of the bacterial family Enterobacteriaceae, increased the proportion of families Enterococcoceae and Streptococcaceae and generally increased ileal microbiota diversity. Conclusively, our data support the idea that natural IgG directly purified from pig plasma and given as a feed supplement can be used in modern swine production as an efficient and cost-effective means for reducing both occurrence of PWD and antibiotics usage and with a potential for the prevention and treatment of other intestinal infectious diseases even if the causative agent might not be known.     

Substrate recognition by the Yersinia type III protein secretion machinery

Type III secretion is the designation given to those protein secretion pathways, primarily in pathogenic Gram-negative bacteria, whose secretion machinery components share an amino acid sequence homology to components of the flagellar basal body. In Yersinia spp., these secretion machineries inject virulence proteins called Yops into the cytosol of target macrophages in an effort to evade phagocytic killing. To date, a clear mechanism by which Yops are recognized by the type III secretion machinery has not been elucidated. Unlike most, if not all, previously characterized protein sorting pathways, the information that identifies Yops as substrates for secretion seems not to be wholly encoded within the Yop peptide sequence. In fact, it appears that at least some of this information is contained within yop mRNAs. This review summarizes recent observations that have been made in this unusual field and proposes models by which proteins may be initiated into this pathway.     

养猪场空气中抗性基因和条件致病菌污染特征

养殖场空气中含有较高浓度的抗生素抗性基因和条件致病菌,对人畜健康具有潜在威胁.采用中流量TSP采样器在某养猪场的生活区、猪舍内和猪舍外3个地点分别采样24 h和48 h,并采集猪舍内的饲料、粪便和饮水添加剂样品.采用普通PCR检测样品中的3类抗生素抗性基因(大环内酯类、β-内酰胺类、四环素类各3个基因)和7种致病菌/条件致病菌基因(弯曲杆菌属、产气荚膜梭菌、肠球菌属、大肠杆菌、小肠结肠炎耶尔森菌、葡萄球菌属和猪链球菌);选取检出率较高的6种基因,采用荧光定量PCR对其浓度进行测定.结果表明: 空气中大环内酯类抗性基因检出了3个,四环素类检出了2个,肠球菌、大肠杆菌、小肠结肠炎耶尔森菌、葡萄球菌等4种条件致病菌在空气样品中和饮水添加剂中都被检测到.绝大部分目的基因的浓度均在10⁴ copies·m^-3以上,并且猪舍附近浓度远高于生活区;猪场内主要的抗生素抗性基因和条件致病菌的可能来源是猪粪便和饮水添加剂.在养猪场内采样24 h即可满足PCR检测要求;在生活区采样48 h的采样效率高于采样24 h,而在猪舍外和猪舍内采样24 h的效率高于采样48 h.     

Intracellular targeting of exoenzyme S of Pseudomonas aeruginosa via type III-dependent translocation induces phagocytosis resistance, cytotoxicity and disruption of actin microfilaments

Exoenzyme S (ExoS) is an ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa . The amino-terminal half of ExoS exhibits homology to the YopE cytotoxin of pathogenic Yersinia . Recently, YopE was found to be translocated into the host cell by a bacteria–cell contact-dependent mechanism involving the ysc -encoded type III secretion system. By using an approach in which exoS was expressed in different strains of Yersinia , including secretion and translocation mutants, we could demonstrate that ExoS was secreted and translocated into HeLa cells by a similar mechanism to that described previously for YopE. Similarly to YopE, the presence of ExoS in the host cell elicited a cytotoxic response, correlating with disruption of the actin microfilament structure. A similar cytotoxic response was also induced by a mutated form of ExoS with a more than 2000-fold reduced ADP-ribosyltransferase activity. However, the enzymatically active ExoS elicited a more definite rounding up of the HeLa cells, which also correlated with decreased viability of the cells after prolonged infection compared with cells infected with strains expressing mutated ExoS or YopE. This suggests that ExoS can act through two different mechanisms on the host cell. The expression of ExoS by Yersinia also mediated an anti-phagocytic effect on macrophages. In addition, we present evidence that extracellularly located P. aeruginosa is able to target ExoS into eukaryotic cells. Taken together, our data suggest that P. aeruginosa , by analogy with Yersinia , targets virulence proteins into the eukaryotic cytosol via a type III secretion-dependent mechanism as part of an anti-phagocytic strategy.     

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. After incubation, free insulin was separated from insulin-anti-insulin antibody complex by treatment with dextran-charcoal. Anti-insulin antibodies in the complex were dissociated from insulin by incubation with 0.23 M HCl and inactivated. The amount of dissociated insulin was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab'-horseradish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was 6.7 pg/assay or 150 ng/liter of serum. The present enzyme immunoassay was 10,000-fold more sensitive than the previously described enzyme immunoassay, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate.     

Characterization ofYersinia enterocolitica sensu stricto

The speciesYersinia enterocolitica is definedsensu stricto on the bases of biochemical and other phenotypic characteristics. Biochemically,Y. enterocolitica contains five major biotypes: 1 through 4 of Niléhn and of Wauters, and the trehalose-negative, metabolically inactive, socalled hare strains in biotype 5 of Niléhn and of Wauters, and biochemically atypical strains, including urease-negative, Simmons' citrate-positive, and lactose-and raffinose-positive strains.Y. enterocolitica sensu stricto was distinguishable from the newly described speciesYersinia kristensenii by sucrose and Voges-Proskauer reactions (negative inY. kristensenii). These species were previously separated by DNA relatedness.Y. enterocolitica was also separable biochemically and by DNA relatedness from the two newly proposed rhamnose-positive species,Yersinia intermedia andYersinia frederiksenii. Strain 161(=CIP 80-27=ATCC 9610) is proposed as the neotype forY. enterocolitica.     

Use of a single procedure for selective enrichment, isolation, and identification of plasmid-bearing virulent Yersinia enterocolitica of various serotypes from pork samples.

Many selective enrichment methods for the isolation of Yersinia enterocolitica from foods have been described. However, no single isolation procedure has been described for the recovery and identification of various plasmid-bearing serotypes. A single improved procedure for selective enrichment, isolation, identification, and maintenance of plasmid-bearing virulent serotypes of Y. enterocolitica from pork samples was developed. Enrichment at 12 degrees C in Trypticase soy broth containing yeast extract, bile salts, and Irgasan was found to be an efficient medium for the recovery of plasmid-bearing virulent strains of Y. enterocolitica representing O:3; O:8; O:TACOMA; O:5, O:27; and O:13 serotypes. MacConkey agar proved to be a reliable medium for the isolation of presumptive colonies, which were subsequently confirmed as plasmid-bearing virulent strains by Congo red binding and low calcium response. Further confirmation by multiplex PCR employed primers directed at the chromosomal ail and plasmid-borne virF genes, which are present only in pathogenic strains. The method was applied to pig slaughterhouse samples and was effective in isolating plasmid-bearing virulent strains of Y. enterocolitica from naturally contaminated porcine tongues. Strains isolated from ground pork and tongue expressed plasmid-associated phenotypes and mouse pathogenicity.     

Divergence among genes encoding the elongation factor Tu of Yersinia Species

Elongation factor Tu (EF-Tu), encoded by tuf genes, carries aminoacyl-tRNA to the ribosome during protein synthesis. Duplicated tuf genes (tufA and tufB), which are commonly found in enterobacterial species, usually coevolve via gene conversion and are very similar to one another. However, sequence analysis of tuf genes in our laboratory has revealed highly divergent copies in 72 strains spanning the genus Yersinia (representing 12 Yersinia species). The levels of intragenomic divergence between tufA and tufB sequences ranged from 8.3 to 16.2% for the genus Yersinia, which is significantly greater than the 0.0 to 3.6% divergence observed for other enterobacterial genera. We further explored tuf gene evolution in Yersinia and other Enterobacteriaceae by performing directed sequencing and phylogenetic analyses. Phylogenetic trees constructed using concatenated tufA and tufB sequences revealed a monophyletic genus Yersinia in the family Enterobacteriaceae. Moreover, Yersinia strains form clades within the genus that mostly correlate with their phenotypic and genetic classifications. These genetic analyses revealed an unusual divergence between Yersinia tufA and tufB sequences, a feature unique among sequenced Enterobacteriaceae and indicative of a genus-wide loss of gene conversion. Furthermore, they provided valuable phylogenetic information for possible reclassification and identification of Yersinia species.     

Biochemical peculiarities of the guinea pig and some possible examples of convergent evolution

Summary The literature was searched for examples of biochemical differences between the guinea pig (Cavia porcellus) and other mammals. Ten well established examples of significant differences were found and are described here. The most striking observation to emerge is that two unusual changes have occurred in the guinea pig (and several other Hystricomorphs) and also in certain New World monkeys, changes that are seen in no other mammalian species examined. The changes are the development of an insulin so divergent in structure that it fails to cross-react with anti-serum to bovine insulin; and the presence of plasma L-asparaginase. The possibility that these are examples of convergent evolution is considered. It is also suggested that the guinea pig may be unique with respect to the number of biochemical differences that it exhibits.Dedicated to Prof. David Shemin in honor of his 70th birthday.