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Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.

Some Escherichia coli strains isolated from intestinal or extraintestinal infections in pigs produce cytotoxic necrotizing factor 1 (CNF1). In order to analyze the role of CNF1 in the pathogenesis of porcine colibacillosis, newborn colostrum-deprived germfree piglets were orally inoculated with a wild-type CNF1-producing strain (M623) or with an isogenic cnf1 mutant (M623DeltaCNF1). The two isogenic strains induced a high mortality with similar lung and serosal inflammatory lesions, indicating that both strains were pathogenic in these piglets. Bacterial counts in various organs of inoculated piglets revealed an intestinal predisposition of M623 and M623DeltaCNF1 strains for the cecum and colon. Extraintestinal organs (lungs, liver, spleen, and kidney) were also colonized by both strains. Similar colonization of intestinal and extraintestinal tissues in animals inoculated with either strain was observed, except in the ileum, where M623 showed a higher colonization than M623DeltaCNF1. Intestinal (ileum and colon), extraintestinal (lung and kidney), and immune (mesenteric lymph nodes and spleen) tissues were sampled at 1 day postinoculation and analyzed for cytokine expression by a reverse transcriptase PCR technique. Inoculation with E. coli M623 induced an enhanced expression of inflammatory cytokines (interleukin-1alpha [IL-1alpha], tumor necrosis factor alpha, and IL-12p40) in the intestinal organs compared to uninoculated piglets or piglets inoculated with nonpathogenic intestinal E. coli 862B, which is also able to colonize the intestinal tract. There was little difference in cytokine transcript levels in the intestinal and extraintestinal organs in piglets inoculated with E. coli strains M623 or M623DeltaCNF1, except in the ileum, where IL-1alpha and IL-8 mRNA levels correlated with bacterial colonization. Expression of regulatory cytokines (gamma interferon and IL-4) was weak in immune tissues from piglets inoculated with M623 or M623DeltaCNF1. Taken together, our data indicate that the CNF1-producing strain, M623, is pathogenic and induces inflammatory cytokine expression in germfree, colostrum-deprived piglets. Nevertheless, in this model, the CNF1 toxin does not appear to be a major factor for pathogenicity or cytokine response, as demonstrated by the use of an isogenic cnf1 mutant.

ABSTRACTSome Escherichia colistrains isolated from intestinal or extraintestinal infections in pigs produce cytotoxic necrotizing factor 1 (CNF1). In order to analyze the role of CNF1 in the pathogenesis of porcine colibacillosis, newborn colostrum-deprived germfree piglets were orally inoculated with a wild-type CNF1-producing strain (M623) or with an isogenic cnf1mutant (M623ΔCNF1). The two isogenic strains induced a high mortality with similar lung and serosal inflammatory lesions, indicating that both strains were pathogenic in these piglets. Bacterial counts in various organs of inoculated piglets revealed an intestinal predisposition of M623 and M623ΔCNF1 strains for the cecum and colon. Extraintestinal organs (lungs, liver, spleen, and kidney) were also colonized by both strains. Similar colonization of intestinal and extraintestinal tissues in animals inoculated with either strain was observed, except in the ileum, where M623 showed a higher colonization than M623ΔCNF1. Intestinal (ileum and colon), extraintestinal (lung and kidney), and immune (mesenteric lymph nodes and spleen) tissues were sampled at 1 day postinoculation and analyzed for cytokine expression by a reverse transcriptase PCR technique. Inoculation with E. coliM623 induced an enhanced expression of inflammatory cytokines (interleukin-1α [IL-1α], tumor necrosis factor α, and IL-12p40) in the intestinal organs compared to uninoculated piglets or piglets inoculated with nonpathogenic intestinal E. coli862B, which is also able to colonize the intestinal tract. There was little difference in cytokine transcript levels in the intestinal and extraintestinal organs in piglets inoculated with E. colistrains M623 or M623ΔCNF1, except in the ileum, where IL-1α and IL-8 mRNA levels correlated with bacterial colonization. Expression of regulatory cytokines (gamma interferon and IL-4) was weak in immune tissues from piglets inoculated with M623 or M623ΔCNF1. Taken together, our data indicate that the CNF1-producing strain, M623, is pathogenic and induces inflammatory cytokine expression in germfree, colostrum-deprived piglets. Nevertheless, in this model, the CNF1 toxin does not appear to be a major factor for pathogenicity or cytokine response, as demonstrated by the use of an isogenic cnf1mutant.

Cytotoxic necrotizing factor types 1 and 2 (CNF1 and -2) produced by pathogenic Escherichia coli strains have 90% conserved residues over 1,014-amino-acid sequences. Both CNFs are able to provoke a remarkable increase in F-actin structures in cultured cells and covalently modify the RhoA small GTPases. In this study, we demonstrated that CNF2 reduced RhoA GTPase activity in the presence and absence of P122(RhoGAP). Subsequently, peptide mapping and amino acid sequencing of CNF2-modified FLAG-RhoA produced in E. coli revealed that CNF2 deamidates Q63 of RhoA-like CNF1. In vitro incubation of the C-terminal domain of CNF2 with FLAG-RhoA resulted also in deamidation of the FLAG-RhoA, suggesting that this region contains the enzymatic domain of CNF2. An oligopeptide antibody (anti-E63) which specifically recognized the altered G-3 domain of the Rho family reacted with glutathione S-transferase (GST)-RhoA and GST-Rac1 but not with GST-Cdc42 when coexpressed with CNF2. In addition, CNF2 selectively induced accumulation of GTP form of FLAG-RhoA and FLAG-Rac1 but not of FLAG-Cdc42 in Cos-7 cells. Taken together, these results indicate that CNF2 preferentially deamidates RhoA Q63 and Rac1 Q61 and constitutively activates these small GTPases in cultured cells. In contrast, anti-E63 reacted with GST-RhoA and GST-Cdc42 but not with GST-Rac1 when coexpressed with CNF1. These results indicate that CNF2 and CNF1 share the same catalytic activity but have distinct substrate specificities, which may reflect their differences in toxic activity in vivo.

The protective capabilities of PVC, natural rubber and milled nitrile rubber gloves currently used in coal liquefaction pilot plants were assessed and the effectiveness of a laundering method was evaluated. The breakthrough times and cumulative permeation for exposure to toluene and to liquefied coal were determined by measurement of radiolabeled phenol tracer in the aqueous receiving medium. Breakthrough times, normalized by division by the square of the material thickness, agreed with the findings of other studies. On exposure to liquefied coal, the nitrile laboratory glove resisted breakthrough the longest (between 12 and 24 hours), although it was the thinnest material tested. Breakthrough times for the other gloves ranged from 2.75 to 6.5 hours with the times for the natural rubber glove materials intermediate to the two PVC glove materials tested. However, when normalized for thickness, both PVC materials were more resistant to breakthrough than rubber. It was found that the laundering method did not completely decontaminate samples of PVC glove material pre-exposed to liquefied coal for 24 hours under laboratory conditions.

Actinobacillus actinomycetemcomitans has been shown to produce a soluble cytotoxic factor(s) distinct from leukotoxin. We have identified in A. actinomycetemcomitans Y4 a cluster of genes encoding a cytolethal distending toxin (CDT). This new member of the CDT family is similar to the CDT produced by Haemophilus ducreyi. The CDT from A. actinomycetemcomitans was produced in Escherichia coli and was able to induce cell distension, growth arrest in G2/M phase, nucleus swelling, and chromatin fragmentation in HeLa cells. The three proteins, CDTA, -B and -C, encoded by the cdt locus were all required for toxin activity. Antiserum raised against recombinant CDTC completely inhibited the cytotoxic activity of culture supernatant and cell homogenate fractions of A. actinomycetemcomitans Y4. These results strongly suggest that the CDT is responsible for the cytotoxic activity present in the culture supernatant and cell homogenate fractions of A. actinomycetemcomitans Y4. This CDT is a new putative virulence factor of A. actinomycetemcomitans and may play a role in the pathogenesis of periodontal diseases.

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A review of fossil mammalian faunas from the Middle Awash indicates they span most of the later Neogene and document evolutionary change in several mammalian groups, especially Primates, Proboscidea and Artiodactyla. Oldowan artefacts first appear in the late Pliocene, while Acheulian and later industries and apparent occupation sites occur in Pleistocene beds.

A specific portion of our environment has been contaminated with Kepone, or chlordecone. Additionally, some specific human exposures to high concentrations of Kepone have been confirmed. Gas chromatography mass spectrometry involving chemical ionization and high resolution mass spectrometry were used to detect, identify and confirm the presence of Kepone, Kepone photoproducts and a reduction product of Kepone in environmental and human samples. Field desorption, field ionization and electron impact mass spectrometric methods, as well as infrared and nuclear magnetic resonance techniques were used to characterize and identify Kepone hydrate and hemiketal in benzene and methanol solutions, respectively.

Thirty-four years ago, the state of Louisiana established a home for lepers and maintained it until Jan. 3, 1921, when it was purchased by the federal government and its operation assumed as a national leprosarium. The records of these two institutions furnish the data from which this paper has been written.Since Dec. 1, 1894, 718 lepers have been admitted (chart 1). Of these, 215 were foreign born (table 1), representing instances in which lepers were admitted to the United States in the incubation, latent or otherwise undiagnosable stages of leprosy.Admissions of native born have numbered 504, with a geographic distribution including all the states of the union except eleven. In table 2 is given the number of cases from each state. This table cannot be taken as a comparative index of the incidence of leprosy in the states because some states have, to a greater extent than others,

On the theory that facts of interest and of possible bearing on the transmission of leprosy might be obtained from a study of the personal histories of a large number of lepers, an investigation has been made of the "individual records" of each leper on his admission to the Culion Leper Colony.The histories, which, of necessity, are very brief, contain, in addition to the name of the individual, the age, sex, nationality, race, birthplace, last place of residence, date of admission to the colony, marital relation, family contact history, probable duration of the disease, and a brief description of the physical condition of the patient at the time of admission.Since a large number of the lepers of the Philippine Islands have only a slight knowledge of the simplest principles of hygiene and sanitation, questions regarding their views

In 1906, Eitner1 observed that the serum of a leper yielded a positive complement-fixation reaction with an extract of a leprous nodule. Two years later,2 he observed similar results with the serum of a second leper, and in addition he noted the occurrence of a positive Wassermann reaction employing an alcoholic extract of guinea-pig heart as antigen. These observations have stimulated a large number of investigations on the subject of complement fixation in leprosy, employing not only the antigens commonly used in the Wassermann reaction, but also extracts of leprous tissues and of various acid-fast bacilli, tuberculin, etc. One result of these investigations has been to spread the impression that in leprosy the Wassermann reaction may be positive in the absence of syphilis, as shown in the following brief review of the literature on the Wassermann reaction in this disease.Meier,3 for example, reported 70 per cent,

After an absence of four months, cholera appeared in the city of Manila in July, 1914. The present epidemic, in which there were reported more than 1,100 cases, suspects and "carriers," did not differ greatly from the ordinary small epidemic. All patients, excepting those who died without medical attention, were treated at San Lazaro Hospital.Of the number mentioned, 330 were genuine cases of cholera, 170 not cholera, and 570 were carriers, so-called; 99 were found dead and sent to San Lazaro morgue for confirmation of diagnosis. The total number of deaths with and without medical attention was 190.The percentage of recoveries among those receiving medical attention at San Lazaro was 72.5.When a patient is admitted to the hospital, a stool specimen is at once taken and sent to the Bureau of Science for bacteriologic examination, and no patient is discharged until at least two successive stool specimens