(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rk-, mk+) phoA supE44 thi-1 gyrA96 relA1 lF-mcrAD(mrr-hsdRMS-mcrBC)W 80dlacZDM15 DlacX74 recA1 endA1 araD139 D(ara, leu)7697 galU galK l- rpsL (StrR) nupG pir-116(DHFR) Strain with T6SSSPI-6 from S. Typhimurium cloned in plasmid R995 Strain with T6SSSPI-19 from S. Gallinarum cloned in plasmid R995 Strain harboring an empty R995 vector Strain containing a derivative of plasmid R995 using a 1,209 bp DNA fragment of T6SSSPI-6 cloned from S. Typhimurium Laboratory collection Laboratory collection This study [42] This study This studySalmonella Typhimurium14028 s MTM753 MTM35 MTM2640 WT/R995 MTM35R MTM35R6 MTM35R19 Plasmids pKD46 pEKA30 pCLF2 pCLF4 pVEX1212 pVEX2212 R995 R995-VC6 R995+SPI-6 R995+SPI-19 doi:ten.1371/journal.pone.0063917.t001 bla PBAD bet exo pSC101 oriTs, AmpR IncQ plasmid that constitutively express Cre recombinase, AmpR Red-swap redesigned vector, CamR Red-swap redesigned vector, KanR Suicide vector harboring a loxP web site followed by a SpR cassette Suicide vector harboring a loxP website followed by a CamR cassette Self-transmissible broad-host range IncP vector A derivative of plasmid R995 with a 1,209 bp DNA fragment of T6SSSPI-6 cloned from S. Typhimurium T6SSSPI-6 cluster from S. Typhimurium 14028 s cloned in vector R995 T6SSSPI-19 cluster from S. Gallinarum 287/91 cloned in vector R995 [43] [44] [50] [50] [44] [44] [44] This study This study [42] Wild-type strain 14028 s DphoN 14028 s DSPI-6 T6SS 14028 s DclpV 14028 s containing an empty R995 vector MTM35 harboring R995 plasmid MTM35 complemented with plasmid R995+SPI-6 MTM35 complemented with plasmid R995+SPI-19 Laboratory collection This study This study This study This study This study This study This studywas assessed in each organ at every time point studied.Modakafusp alfa No differences have been observed on colony forming units (CFU) indicating that R995 and its derivatives are highly stable in vivo.D-chiro-Inositol samples were stained with hematoxylin and eosin and ten fields per sample were examined and scored by a educated veterinary pathologist to decide histopathological changes.PMID:23509865 Experimental Infections of ChickensS. Typhimurium strains were grown aerobically at 42uC for 16 hours in LB broth. This temperature of incubation was used since it corresponds for the body temperature of chicks. For single and competitive infections, fifteen 4-day old unsexed White Leghorn chicks were orally inoculated with 109 CFU of a single strain or with an equal mixture on the strains to be tested inside a volume of 100 ml of sterile PBS. The inoculum was serially diluted and plated to determine the titer and input ratio. Five birds from the infected group had been sacrificed by asphyxiation with CO2 on days 1, 3 and 9 post-infection. Ileum, cecum (including contents), liver and spleen had been collected. These organs have been homogenized in sterile PBS and serial ten-fold dilutions spread on LB agar plates containing the appropriate antibiotics for determination of CFU. For histopathological evaluation, the cecum and liver of experimental animals were fixed in ten formalin for 24 h followed by incubation in 70 ethanol after which embedded in paraffin. TheStatistical AnalysisData obtained from competitive infection experiments have been calculated as a imply ratio of logarithmically converted CFU of mutant to wild form normalized for the input ratio. Error bars indicate common error. Statistical significance was determined working with a two-tailed Students t-test. P values of ,0.05 had been considered statisti.
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