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COMP.EMULATORS.MISC Frequently Asked Questions List Version: 1.1.1 (1997-Jan-14) comp.emulators.misc Frequently Asked Questions ########################################################################## # Copyright 1995, 1996, 1997 Adam Roach # # You may distribute this document freely under the conditions that it is # transmitted to all parties (1) in its entirety, (2) unmodified, and # (3) free of charge. It is explicitly stated that this document MAY NOT # be included in any off-line compilations for which any remuneration is # expected without prior written permission of the copyright holder. # # Web-accessible versions of this document may be made available only # if they are updated automatically from one of the following sources # no less frequently than once per month: # - The semi-monthly posting to news.groups # - The FAQ archive at rtfm.mit.edu # - The web pages found at # # Permission to create derivative works may be granted on a per-case # basis. E-mail me at the address below if you wish to create such works.
H8 Emulation W H19 Now Available For Mac Pro
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FIRE Emulator for H8S and H8/300H 6 ©1989-2018 Lauterbach GmbH Quick Start Before debugging can be started, the emulator must be configured by software: Ready-to-run setup files for most standard compilers can be found in the folder ~~/demo/h8s/compiler. All setup files are designed to run the emulator stand alone without target hardware.
Abstract Shiga toxin-producing Escherichia coli (STEC), responsible for the hemolytic uremic syndrome, is an endemic pathogen in Argentina. We studied the prevalence of STEC in fecal samples from cats and dogs of Buenos Aires city and suburbs. Cultures were used for screening stx1/stx2 and rfbO157 by multiplex PCR. Coli-positive colonies for these genes were further characterized for the eae gene and for serotypes. In dogs, 17 (3.7%), 19 (4.2%) and 34 (7.5%) of samples were positive for stx2, stx1 and rfb, respectively. In cats, six (4.0%) of the samples were positive for stx2, three (2.0%) for stx1 and four (2.7%) for rfbO157. In 18 (4.0%) of the dog samples, a bacteriological diagnosis was obtained by isolation.
The percentage of positive isolates corresponding to the rfbO157 and to the stx2 genotypes were 2.9% and 1.1%, respectively. In four of the cat samples, the bacteriological diagnosis for stx2 (2.6% prevalence of STEC) was confirmed. Although these data suggest that the high infection index of STEC in children in Argentina does not seem to be due mainly to the role of cats and dogs, there are some strains with virulence genes in common for humans and their domestic animals. Multiplex PCR amplification: lanes 1–4, amplification of rfb0157-positive isolates; lane 5, amplification of Escherichia coli EDL 933 (O157:H7, stx1/stx2, eae) template as a positive control; lanes 6 100 bp ladder, lane 7–11 amplification of stx2-positive isolates, and lane 12, negative control. Molecular sizes are indicated on the left.
From samples compatible with any of the mentioned genes in the confluence zone, the presence of the genes was investigated in up to 300 CFU. All colonies positive to any of the genes under study were further identified as E. Coli after Gram staining, oxidase and catalase tests, subculturing in triple sugar iron agar (TSI) and determining biochemical characteristics by standard methods. The presence of the eae gene was searched by PCR in all E.
Coli isolates carrying stx1/stx2 and/or rfb O157 sequences as follows: eae PCR The primers reported by, which amplify a 346 bp-fragment of the conserved region of the eae gene that encodes for the intimin ( eae1: 5′-GGAACGGCAGAGGTTAATCTGCAG; eae2: 5′-GGCGCTCATCATAGTCTTTC), were used. The assay conditions were as follows: the 50 µL-PCR mixture contained 0.3 µM (each) of the two eae-specific primers, 200 µM of each dNTP 2 mM MgCl 2 and 1 U of Taq Platinum (Invitrogen) DNA polymerase in 1 × buffer according to the manufacturer's instructions. The amplification program utilized was denaturation at 94°C for 5 min, followed by 30 cycles at 94°C for 45 s, at 62°C for 30 s, at 72°C for 30 s, completing it with a final extension at 72°C for 7 min and maintaining it at 4°C until analysis. Detection limit Serial dilutions in base 10 were performed in saline solution from a culture from the positive control strain ( E. Coli EDL 933), and the bacterial load was determined by counting the CFU in solid media. Three dogs identified by the same multiplex PCR as the negatives were used for these assays.
From each animal, rectal swab samples were collected in duplicate. One of the duplicate swabs was processed as a negative control, and the other was inoculated with 10 µL of a control strain dilution and was processed in parallel. All the swabs were pre-enriched and cultivated in Mac Conkey agar and Mac Conkey Sorbitol-CT agar.
Multiplex PCR was performed in the confluence zone as described. The detection limit of the screening system was determined as the higher culture dilution that was detected by multiplex PCR.
These assays from the rectal swab were reproduced three times with each serial dilution. Serotyping Identification of somatic (O) and flagelar (H) antigens was carried out following the standard methods and using currently available O (O1 to O181) and H (H1 to H56) antisera as described elsewhere.
Analysis of the collected data The data are expressed as prevalence or proportion and its respective 95% of confidence interval. The differences in prevalence according to sex, age and presence of diarrhea both at the moment of the sampling and a month before collection were analyzed by means of the χ 2 and Fischer tests.
Results The detection limit of the system was 20 CFU contained in 10 µL of microbial suspension, inoculated in a negative recto-anal swabbing, kept in Stuart medium and submitted to the same enrichment and plating in Mac Conkey and Mac Conkey Sorbitol-CT agar as for the samples. The limit was established as the maximal dilution allowing the observation of the 3 amplicons. In some samples, amplification inhibitors (especially those of stx2) were observed (data not shown). When serial dilutions of pure culture E. Coli EDL 933 were performed in water, a detection limit of 6.6 10 3 was observed (data not shown). Seventy (15.5%) of the dog samples and 13 (8.7%) of the cat samples were positive to the screening for one of the genes evaluated from the confluence zone. Through the formation of pools of individual colonies and screening by multiplex PCR, 22 cases resulted in a bacteriological diagnosis of certainty of strains that were identified as being positive to one of the studied genes.
Serotypes Genetic markers Total number of positive samples Total number of strains isolated Diarrheagenic category Dogs O91:H16 stx2 1 1 STEC O91:H21 stx2 1 2 STEC O157:H − stx2 1 1 STEC O157:H16 eae, rfbO157 9 34 AEEC O157:H16 rfbO157 1 1 – O157:H29 eae, rfbO157 1 1 – O157:H29 rfbO157 1 1 – O157:H45 eae, rfbO157 1 1 AEEC O178:H19 stx2 1 5 STEC O178:H19 stx2, eae 1 1 STEC/EHEC ONT:H19 stx2 1 1 STEC OR:H43 rfbo157 1 1 – Cats O22:H − stx2 1 1 STEC O22:H8 stx2 1 1 STEC ONT:H8 stx2 1 1 STEC ONT:H19 stx2 2 1 STEC. Serotypes Genetic markers Total number of positive samples Total number of strains isolated Diarrheagenic category Dogs O91:H16 stx2 1 1 STEC O91:H21 stx2 1 2 STEC O157:H − stx2 1 1 STEC O157:H16 eae, rfbO157 9 34 AEEC O157:H16 rfbO157 1 1 – O157:H29 eae, rfbO157 1 1 – O157:H29 rfbO157 1 1 – O157:H45 eae, rfbO157 1 1 AEEC O178:H19 stx2 1 5 STEC O178:H19 stx2, eae 1 1 STEC/EHEC ONT:H19 stx2 1 1 STEC OR:H43 rfbo157 1 1 – Cats O22:H − stx2 1 1 STEC O22:H8 stx2 1 1 STEC ONT:H8 stx2 1 1 STEC ONT:H19 stx2 2 1 STEC. Serotypes Genetic markers Total number of positive samples Total number of strains isolated Diarrheagenic category Dogs O91:H16 stx2 1 1 STEC O91:H21 stx2 1 2 STEC O157:H − stx2 1 1 STEC O157:H16 eae, rfbO157 9 34 AEEC O157:H16 rfbO157 1 1 – O157:H29 eae, rfbO157 1 1 – O157:H29 rfbO157 1 1 – O157:H45 eae, rfbO157 1 1 AEEC O178:H19 stx2 1 5 STEC O178:H19 stx2, eae 1 1 STEC/EHEC ONT:H19 stx2 1 1 STEC OR:H43 rfbo157 1 1 – Cats O22:H − stx2 1 1 STEC O22:H8 stx2 1 1 STEC ONT:H8 stx2 1 1 STEC ONT:H19 stx2 2 1 STEC. Serotypes Genetic markers Total number of positive samples Total number of strains isolated Diarrheagenic category Dogs O91:H16 stx2 1 1 STEC O91:H21 stx2 1 2 STEC O157:H − stx2 1 1 STEC O157:H16 eae, rfbO157 9 34 AEEC O157:H16 rfbO157 1 1 – O157:H29 eae, rfbO157 1 1 – O157:H29 rfbO157 1 1 – O157:H45 eae, rfbO157 1 1 AEEC O178:H19 stx2 1 5 STEC O178:H19 stx2, eae 1 1 STEC/EHEC ONT:H19 stx2 1 1 STEC OR:H43 rfbo157 1 1 – Cats O22:H − stx2 1 1 STEC O22:H8 stx2 1 1 STEC ONT:H8 stx2 1 1 STEC ONT:H19 stx2 2 1 STEC.